WO2020263782A1 - Méthodes et compositions permettant de modifier des acides nucléiques et de tuer des cellules - Google Patents

Méthodes et compositions permettant de modifier des acides nucléiques et de tuer des cellules Download PDF

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WO2020263782A1
WO2020263782A1 PCT/US2020/039063 US2020039063W WO2020263782A1 WO 2020263782 A1 WO2020263782 A1 WO 2020263782A1 US 2020039063 W US2020039063 W US 2020039063W WO 2020263782 A1 WO2020263782 A1 WO 2020263782A1
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polymer
nucleic acid
crispr
mol
alkoxylene
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Rodolphe Barrangou
Matthew A. NETHERY
Claudio Hidalgo-Cantabrana
Paul Bernasconi
Marianela RODRIGUEZ
Melissa Ann LAMSON
Frank Reinhold
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BASF Plant Science Co GmbH
North Carolina State University
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BASF Plant Science Co GmbH
North Carolina State University
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention relates generally to methods of modifying the genome of an organism comprising contacting genome of the organism with a composition comprising a CRISPR-Cas nucleic acid and a polyalkyleneimine-alkoxylene polymer such as a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer.
  • a composition comprising a CRISPR-Cas nucleic acid and a polyalkyleneimine-alkoxylene polymer such as a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer.
  • PEI polyethyleneimine
  • PEO polyethylene oxide
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • cas associated sequences
  • CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and phages, as novel“spacers.”
  • CRISPR-Cas systems consist of arrays of short DNA repeats interspaced by hypervariable sequences, flanked by cas genes, that provide adaptive immunity against invasive genetic elements such as phage and plasmids, through sequence-specific targeting and interference (Barrangou et al. 2007. Science. 315: 1709-1712; Brouns et al. 2008. Science 321 :960-4; Horvath and Barrangou. 2010. Science. 327: 167-70; Marraffmi and Sontheimer. 2008. Science. 322: 1843-1845; Bhaya et al. 2011. Annu. Rev. Genet. 45:273-297; Terns and Terns. 2011. Curr. Opin. Microbiol.
  • RNA. 4:267-278 invasive DNA sequences are acquired as novel“spacers” (Barrangou et al. 2007. Science. 315: 1709-1712), each paired with a CRISPR repeat and inserted as a novel repeat-spacer unit in the CRISPR locus. Subsequently, the repeat-spacer array is transcribed as a long pre-CRISPR RNA (pre-crRNA) (Brouns et al. 2008. Science 321 :960-4), which is processed into small interfering CRISPR RNAs (crRNAs) that drive sequence-specific recognition.
  • pre-crRNA pre-CRISPR RNA
  • crRNAs guide nucleases towards complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases (Garneau et al. 2010. Nature. 468:67-71; Haurwitz et al. 2010. Science. 329: 1355-1358; Sapranauskas et al. 2011. Nucleic Acid Res. 39:9275-9282; Jinek et al. 2012. Science. 337:816-821; Gasiunas et al. 2012. Proc. Natl. Acad. Sci. 109:E2579-E2586; Magadan et al. 2012. PLoS One. 7:e40913; Karvelis et al. 2013. RNA Biol.
  • the specialized Cas endonucleases process the pre-crRNAs, which then assemble into a large multi-Cas protein complex capable of recognizing and cleaving nucleic acids complementary to the crRNA.
  • a different process is involved in Type II CRISPR-Cas systems.
  • the pre-CRNAs are processed by a mechanism in which a trans-activating crRNA (tracrRNA) hybridizes to repeat regions of the crRNA.
  • the hybridized crRNA-tracrRNA are cleaved by RNase III and following a second event that removes the 5’ end of each spacer, mature crRNAs are produced that remain associated with the both the tracrRNA and Cas9.
  • the mature complex locates a target dsDNA sequence (‘protospacer’ sequence) that is complementary to the spacer sequence in the complex and cuts both strands.
  • Target recognition and cleavage by the complex in the Type II system not only requires a sequence that is complementary between the spacer sequence on the crRNA- tracrRNA complex and the target‘protospacer’ sequence but also requires a protospacer adjacent motif (PAM) sequence located at the 3’ end of the protospacer sequence.
  • PAM protospacer adjacent motif
  • Embodiments of the invention provide methods of editing/modifying a target nucleic acid (e.g., target region; target DNA/RNA) in the genome of a cell (e.g., a target cell; host cell) of an organism, comprising introducing into the cell a first composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer) and a CRISPR-Cas array (CRISPR array, crRNA, crDNA), wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to a target nucleic acid in the genome of the cell, thereby editing/modifying the target nucleic acid in the genome of the cell/organism.
  • a target nucleic acid e.g., target region; target DNA/RNA
  • a cell e.g., a target cell; host cell
  • a first composition comprising a first polyalkyleneimine-
  • Embodiments of the invention provide methods for site-specific cleavage of target nucleic acid (e.g., target region; target DNA/RNA) in the genome of a cell (e.g., a target cell; host cell) of an organism, comprising introducing into the cell a first composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer) and a CRISPR-Cas array (CRISPR array, crRNA, crDNA), wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to a target nucleic acid in the genome of the cell, thereby producing a site specific cleavage in a region defined by complementary binding of the spacer sequence of the CRISPR array to the target nucleic acid in the genome of the cell/organism.
  • a first polyalkyleneimine-alkoxylene polymer e.g., a polyethyleneimine
  • Embodiments of the invention provide methods for transcriptional control of a target nucleic acid (e.g., target region; target DNA/RNA) in the genome of a cell (e.g., a target cell; host cell) of an organism, comprising introducing into the cell a composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer), a Type II CRISPR-Cas array (CRISPR array), and a nucleic acid encoding a deactivated Cas9 nuclease, wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to the target nucleic acid in the genome of the cell, thereby controlling the transcription of the target nucleic acid in the genome of the cell/organism.
  • a target nucleic acid e.g., target region; target DNA/RNA
  • a cell e.g., a target cell; host
  • Embodiments of the invention provide methods for transcriptional control of a target nucleic acid (e.g., target region; target DNA/RNA) in the genome of a cell (e.g., a target cell; host cell) of an organism, comprising introducing into the cell a composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer), a Type I CRISPR-Cas array (CRISPR array, crRNA, crDNA), and a nucleic acid encoding a deactivated Cas3, wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to the target nucleic acid in the genome of the cell, thereby controlling the transcription of the target nucleic acid.
  • a target nucleic acid e.g., target region; target DNA/RNA
  • a cell e.g., a target cell; host cell
  • a composition comprising a
  • Embodiments of the invention provide methods of killing a cell (e.g., a target cell; host cell) of an organism, comprising introducing into the cell a first composition comprising a first polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer and a CRISPR-Cas array (CRISPR array, crRNA, crDNA), wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to a target nucleic acid (e.g., target region; target DNA/RNA) in the genome of the cell, thereby killing the cell.
  • a target nucleic acid e.g., target region; target DNA/RNA
  • Fig. 2 shows chemical-based delivery of DNA using polymers 1, 3, 7, and 9.
  • Plasmid DNA packaged within each polymer, encoding enhanced green fluorescent protein (eGFP) can be delivered by mixing with wheat protoplast cells.
  • Micrographs depicting representative GFP signals were taken two days post transfection.
  • As a positive control cells were transfected with the GFP reporter plasmid in the presence of polyethylene glycol (PEG).
  • Transfection efficiencies were calculated by counting the number of GFP expressing cells and dividing by the number of living cells. Data presented are means of three independent experiments.
  • Fig. 3A-3B shows transfection of the cells using polymers of the invention.
  • Fig. 3A shows transformation efficiency using different amounts of DNA.
  • Fig. 3B shows transformation efficiencies using different amounts of polymer (polymer 9). Transfection efficiencies were calculated by counting the number of GFP expressing cells and dividing by the number of living cells. Data presented are means of two independent experiments. Photographs depicting representative efficiencies were taken two days post transfection. Positive control cells were transfected using polyethylene glycol and negative controls were treated with plasmid DNA only.
  • the transitional phrase consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. See, In re Herz , 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03.
  • the term consisting essentially of as used herein should not be interpreted as equivalent to comprising.
  • “about X,” where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measureable value may include any other range and/or individual value therein.
  • nucleic acid refers to RNA or DNA that is linear, circular or branched, single or double stranded, or a hybrid thereof.
  • the terms also encompass RNA/DNA hybrids.
  • the RNA and/or DNA can include a chemically modified base such as those that are not usually found in nature.
  • dsRNA When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others may be used such as for antisense, dsRNA, and ribozyme pairing.
  • polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.
  • Other modifications such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.
  • nucleic acid sequence As used herein, the terms“polynucleotide” “nucleotide sequence,”“nucleic acid molecule” and“nucleic acid sequence” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA and/or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded and/or double stranded.
  • nucleotide sequence nucleic acid, nucleic acid molecule, oligonucleotide and polynucleotide may also be used to refer to a heteropolymer of nucleotides.
  • Nucleic acid molecules and/or nucleotide sequences provided herein are presented in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.
  • a 5' region as used herein refers to the region of a polynucleotide that is nearest the 5' end.
  • an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
  • a 3' region as used herein refers to the region of a polynucleotide that is nearest the 3' end.
  • an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
  • the term“gene” refers to a nucleic acid molecule capable of being used to produce mRNA, tRNA, rRNA, miRNA, anti-microRNA, regulatory RNA, and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions). A gene may be isolated by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • a synthetic nucleic acid or nucleotide sequence refers to a nucleic acid or nucleotide sequence that is not found in nature but is constructed by human intervention and as a consequence is not a product of nature.
  • Alkyl as used herein alone or as part of another group refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms, which can be referred to as a Cl- C20 alkyl.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n- decyl, and the like.
  • Loweralkyl as used herein, is a subset of alkyl, and, in some embodiments, refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
  • Representative examples of loweralkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like.
  • alkyl or loweralkyl is intended to include both substituted and unsubstituted alkyl or loweralkyl unless otherwise indicated and these groups may be substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(0) m , haloalkyl-S(0) m , alkenyl-S(0) m , alkynyl-S(0) m , cycloalkyl
  • Alkenyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkenyl 1 to 4 carbon atoms) and includes 1 to 8 double bonds in the normal chain, and can be referred to as a C1-C20 alkenyl.
  • Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4- heptadiene, and the like.
  • alkenyl or loweralkenyl is intended to include both substituted and unsubstituted alkenyl or loweralkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and loweralkyl above.
  • Alkynyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include at least 1 triple bond in the normal chain, and can be referred to as a C1-C20 alkynyl.
  • Representative examples of alkynyl include, but are not limited to, 2- propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like.
  • alkynyl or loweralkynyl is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
  • Halo as used herein refers to any suitable halogen, including -F, -Cl, -Br, and -I.
  • Mercapto as used herein refers to an -SH group.
  • Cyano as used herein refers to a -CN group.
  • Hydroxyl as used herein refers to an -OH group.
  • Nitro as used herein refers to an -NO2 group.
  • Alkoxy as used herein alone or as part of another group refers to an alkyl or loweralkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -0-.
  • alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
  • Acyl as used herein alone or as part of another group refers to a -C(0)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.
  • Haloalkyl as used herein alone or as part of another group refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.
  • Aryl as used herein alone or as part of another group refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings.
  • Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • the term aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
  • an aryl is substituted with an alkyl, alkenyl, and/or alkynyl.
  • Arylalkyl as used herein alone or as part of another group refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.
  • arylalkenyl and arylalkynyl as used herein alone or as part of another group refer to an aryl group, as defined herein, appended to the parent molecular moiety through an alkenyl group and alkynyl, respectively, each as defined herein.
  • Amino as used herein means the radical -NH 2.
  • Alkylamino as used herein alone or as part of another group means the radical -NHR, where R is an alkyl group.
  • Ester as used herein alone or as part of another group refers to a -C(0)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • Amide as used herein alone or as part of another group refers to a -C(0)NR a R b radical, where R a and R 3 ⁇ 4 are any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • Sulfonamide as used herein alone or as part of another group refers to a -S(0) 2 NR a R- b radical, where R a and 3 ⁇ 4 are any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.
  • the terms increase, increases, increased, increasing, enhance, and similar terms indicate an elevation in the specified parameter of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more unless otherwise specifically noted within the text.
  • the terms“reduce,”“reduced,”“reducing,”“reduction,”“diminish,” “suppress,” and“decrease” and similar terms refer to a decrease in the specific parameter of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% unless otherwise specifically noted within the text.
  • compositions comprising a polyalkyleneimine-alkoxylene polymer and a nucleic acid molecule.
  • a polyalkyleneimine-alkoxylene polymer of the present invention may also be referred to interchangeably herein as an alkoxylated polyethyleneimine.
  • the polyalkyleneimine is a polyethyleneimine and/or a polypropyleneimine.
  • the alkoxylene is one or more ethylene oxide, propylene oxide, and/or butylene oxide unit(s).
  • compositions comprising a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer and a nucleic acid molecule.
  • PEI-PEO may be referenced herein as an example, but other polyalkyleneimine-alkoxylene polymers within the scope of this invention may be used.
  • the polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • the composition may comprise and/or be in an aqueous solution.
  • the composition comprises and/or is a complex of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and at least one nucleic acid molecule.
  • a complex and grammatical variations thereof as used herein refer to a molecular entity comprising at least two components that are associated with one another.
  • the two or more components may be different (e.g., a PEI-PEO polymer and a nucleic acid) and/or one or more of the components may be charged (e.g., ionic) or uncharged.
  • a complex may be formed by covalent, non-covalent, and/or electrostatic interactions between the two or more components.
  • a complex comprises at least two different components that are covalently bonded together.
  • a complex comprises at least two different components that are associated with one another via electrostatic interaction(s).
  • a nucleic acid molecule is electrostatically bound and/or covalently bound to a portion of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a composition of the present invention may comprise one or more (e.g., 1, 2, 5, 10, 50, 100, 1,000, 5,000, 10,000, or more) nucleic acid molecule(s) and/or one or more (e.g., 1, 2, 5, 10, 50, 100, 1,000, 5,000, 10,000, or more) polyalkyleneimine-alkoxylene polymer(s).
  • the composition comprises two or more nucleic acid molecules, which may be complexed with a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a single polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) is complexed with two or more nucleic acid molecules.
  • a single nucleic acid molecule is complexed with two or more polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers).
  • a nucleic acid molecule may have any suitable length.
  • a nucleic acid molecule has a length that is not prohibitive of being delivered and/or transformed into a host cell.
  • a nucleic acid molecule has a length of about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 bases to about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilobases in length.
  • a nucleic acid molecule useful with this invention may be any nucleic acid or combination of nucleic acids (e.g, DNA, RNA, or any combination thereof) of interest for transforming the cell of an organism.
  • the nucleic acid molecule may be plasmid DNA, genomic DNA, plastid DNA, mitochondrial DNA, phage DNA, cDNA, mRNA, siRNA, shRNA, miRNA, piRNA, synthetic DNA, PCR amplicons, and/or antisense nucleic acid or any combination thereof.
  • the nucleic acid molecule may be a negatively charged peptide nucleic acid (PNA).
  • the nucleic acid may be any coding or noncoding nucleic acid (e.g., any transcribed and/or non-transcribed nucleic acids).
  • a nucleic acid molecule useful with this invention may be a nucleic acid molecule that is heterologous to the organism into which it is being introduced (e.g., a heterologous nucleic acid molecule or a heterologous polynucleotide) or it may be heterologous with regard to the position in the genome into which it is introduced.
  • heterologous refers to a nucleic acid molecule or nucleotide sequence that either originates from another species or is from the same species or organism but is modified from either its original form or its original position in the genome, or the form primarily expressed in the cell.
  • a polynucleotide derived from an organism or species different from that of the cell into which the polynucleotide is introduced is heterologous with respect to that cell and the cell's descendants.
  • a heterologous polynucleotide includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule.
  • a nucleic acid molecule may comprise or be comprised in a vector, a nucleic acid construct, an expression cassette, and/or a plasmid.
  • a vector, a nucleic acid construct, an expression cassette, and/or a plasmid may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • a vector, a nucleic acid construct, an expression cassette, and/or a plasmid may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • Nucleic acid molecules/nucleic acid constructs, expression cassettes, plasmids and/or vectors useful with this invention may be included in a composition with a polymer of the present invention.
  • the nucleic acid molecules/nucleic acid constructs, expression cassettes, plasmids and/or vectors may be complexed with polymers of the present invention.
  • expression cassette means a heterologous nucleic acid construct comprising a nucleotide sequence of interest (e.g., the nucleic acid molecules/constructs of the invention (e.g., a synthetic tracr nucleic acid, a synthetic CRISPR nucleic acid, a synthetic CRISPR array, a chimeric nucleic acid construct; a nucleotide sequence encoding a polypeptide of interest, a Type I polypeptide, Type II polypeptide, Type III polypeptide, Type IV polypeptide, Type V polypeptide and/or Type VI polypeptide), wherein said nucleotide sequence of interest is operably associated with at least a control sequence (e.g., a promoter).
  • a control sequence e.g., a promoter
  • nucleic acid molecule, polynucleotide or nucleic acid construct of the present invention is generally free of nucleotide sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was derived (such as coding sequences present at the 5' or 3' ends).
  • nucleic acid molecule of this invention can include some additional bases or moieties that do not deleteriously or materially affect the basic structural and/or functional characteristics of the nucleic acid molecule.
  • an isolated nucleic acid molecule or isolated nucleotide sequence is a nucleic acid molecule or nucleotide sequence that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Accordingly, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • 5' non-coding e.g., promoter
  • the term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid nucleic acid molecule encoding an additional polypeptide or peptide sequence.
  • isolated can further refer to a nucleic acid molecule, polynucleotide, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and/or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized).
  • an isolated fragment is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found as such in the natural state. Isolated does not mean that the preparation is technically pure (homogeneous), but rather can be sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • an isolated nucleic acid molecule, nucleotide sequence, and/or polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure (w/w) or more.
  • an isolated nucleic acid, nucleotide sequence, and/or polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or more enrichment of the nucleic acid (w/w) is achieved as compared with the starting material.
  • a nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer may be present in a composition in a weight ratio of about 1 : 10 to about 1 :600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer), or any range or value therein.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:10, 1:25, 1:50, 1:75, 1:100, 1:250, 1:500, 1:750, 1:1,000, 1:2,000, 1:3,000, 1:4,000, 1:5,000, 1:6,000, 1:7,000, 1:8,000, 1:9,000, 1:10,000, 1:15,000, 1:20,000, 1:25,000, 1:30,000, 1:35,000, 1:40,000, 1:45,000, 1:50,000, 1:55,000, 1:60,000, 1:70,000, 1:80,000, 1:90,000, 1:100,000, 1:200,000, 1:300,000, 1:400,000, 1:500,000 or 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer), or any range or value therein.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:10 to about 1:100, about 1:100 to about 1:1,000, about 1:1,000 to about 1:5,000, about 1:5,000 to about 1:10,000, about 1:5,000 to about 1:20,000, about 1:5,000 to about 1:50,000, about 1:5,000 to about 1:100,000, about 1:5,000 to about 1:200,000, about 1:5,000 to about 1:500,000, about 1:5,000 to about 1:600,000, about 1:10,000 to about 1:100,000, about 1:10,000 to about 1:600,000, about 1:30,000 to about 1:50,000, about 1:10,000 to about 1:50,000, about 1:200,000 to about 1:400,000, about 1:50,000 to about 1:500,000 or about 1:50,000 to about 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer).
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:5000 to about 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer).
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:10,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:20,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:100,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:600,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:5000 (nucleic acid molecule polyalkyleneimine-alkoxylene polymer).
  • a nucleic acid molecule is present in a composition of the invention in an amount of about 0.1 nanograms per milligram of the polyalkyleneimine- alkoxylene polymer to about 400 ng/mg of the polyalkyleneimine-alkoxylene polymer or more.
  • a nucleic acid molecule is present in a composition of the invention in an amount of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 nanograms per milligram of the polyalkyleneimine-alkoxylene polymer to about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 or more nanograms per milligram of the polyalkyleneimine- alkoxylene polymer.
  • Example compositions of the invention may comprise a nucleic acid molecule and polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) with the nucleic acid molecule present in an amount relative to 30 mg polymer as set forth in Table 1.
  • polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • about 100 mg PEI-PEO polymer may combined with 100 m ⁇ water to achieve a final volume of about 200 m ⁇ .
  • about 60 m ⁇ of polymer may be combined with DNA.
  • about 60 m ⁇ of this solution comprises about 30 mg of polymer.
  • a ratio of about 10 ng plasmid to 1 mg PEI-PEO polymer is achieved.
  • 1000 ng of plasmid DNA may be added to 30 mg of polymer (60 m ⁇ solution) to achieve ratio of about 33.3 ng plasmid to 1 mg PEI-PEO polymer.
  • 1500 ng or 6000 ng of plasmid DNA may be added to 30 mg of polymer (60 m ⁇ solution), then the ratio that is achieved is about 50 ng plasmid in 1 mg PEI-PEO polymer or about 200 ng plasmid in 1 mg PEI-PEO polymer, respectively.
  • a polyalkyleneimine (e.g., PEI) polymer, polyalkoxylene (e.g., PEO) polymer and/or polyalkyleneimine-alkoxylene (e.g., PEI-PEO) polymer of the present invention may be and/or may be prepared as described in U.S. Patent Nos. 7,736,525 and 9,068,147 and/or International Publication No. WO 2017/102556, the contents of each of which are incorporated herein by reference in their entirety.
  • a polyalkyleneimine- alkoxylene polymer may be cationic.
  • a polyalkyleneimine-alkoxylene polymer has an overall cationic charge, but a portion of the polyalkyleneimine-alkoxylene polymer is nonionic and/or anionic, which may reduce the overall charge per weight of the polymer.
  • a portion of the polyalkyleneimine-alkoxylene polymer e.g., an outer portion and/or shell
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may have a zeta potential of at least about +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, or more, optionally when present in a composition having a pH of about 7 and/or measured using methods known to those of skill in the art.
  • a polyalkyleneimine-alkoxylene polymer e.g., a non-quatemized or quatemized PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer of the present invention is water soluble, optionally at room temperature.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may have a PEI backbone and one or more alkoxylene unit(s) (e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)) substituted at one or more places (e.g., at one or more nitrogens) along the PEI backbone.
  • alkoxylene unit(s) e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)
  • the polyalkyleneimine-alkoxylene polymer of the present invention may have a polypropyleneimine backbone and one or more alkoxylene unit(s) (e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)) substituted at one or more places (e.g., at one or more nitrogens) along the polypropyleneimine backbone.
  • An alkoxylene unit may replace a hydrogen atom in the PEI or polypropyleneimine backbone (e.g., a hydrogen atom attached to a primary amine, secondary amine, and/or tertiary amine in the PEI backbone).
  • a hydrogen atom in the PEI or polypropyleneimine backbone (e.g., a hydrogen atom attached to a primary amine, secondary amine, and/or tertiary amine in the PEI backbone) is replaced with a single alkoxylene unit (i.e., a monoalkoxylene) or with a polyalkoxylene chain (e.g., a moiety comprising two or more alkoxylene units such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a monoalkoxylene or a polyalkoxylene chain having 2 or 3 to 4, 5, 6, or 8 alkoxylene units (e.g., ethylene oxide units).
  • a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a polyalkoxylene chain having 8, 9, or 10 to 11, 12, or 13 alkoxylene units (e.g., ethylene oxide units).
  • a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a polyalkoxylene chain having 16, 17, or 18 to 19, 20, 21, or 22 alkoxylene units (e.g., ethylene oxide units).
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may be a core-shell polymer.
  • the core comprises an interior portion of the polymer and the shell comprises an outer portion of the polymer.
  • the shell comprises end or terminal portions of a polymer chain.
  • the core comprises a portion or all of the PEI or polypropyleneimine of the polyalkyleneimine-alkoxylene polymer.
  • the core (e.g., PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer) may have a diameter of about 1, 2, 4, 6, or 8 to about 10, 20, 30, 40, 50, 60, 70, or 80 nm such as about 1 nm to about 10 nm, about 5 nm to about 15 nm, about 20 to about 50 nm, 40 nm to about 80 nm, or 60 nm to about 70 nm.
  • the core has a diameter of about 5, 10, or 65 nm.
  • the core comprises a portion or all of the PEI of a PEI-PEO polymer.
  • the shell comprises a portion or all of the alkoxylene (e.g., PEO) of the polyalkyleneimine- alkoxylene polymer.
  • the core comprises a portion or all of the PEI of a PEI-PEO and the shell comprises a portion or all of the PEO of the PEI-PEO polymer.
  • a shell and/or outer surface comprising alkoxylene may aid in controlling and/or reducing the toxicity of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) to a cell and/or may block and/or reduce exposure of charged moieties (e.g., cationic moieties) on the polymer.
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a particle e.g., a microparticle or nanoparticle
  • a polyalkyleneimine-alkoxylene polymer may comprise a number of alkoxylene units, such as ethylene oxide units, per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 1, 2, or 3 to about 4, 5, 6, or 8 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, about 8, 9, or 10 to about 11, 12, or 13 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, or about 16, 17, or 18 to about 19, 20, 21, or 22 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer.
  • the number of alkoxylene units may be determined per primary amine of PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a primary amine as used herein in reference to PEI or polypropyleneimine in a polyalkyleneimine-alkoxylene polymer refers to PEI or polypropyleneimine prior to formation of the polyalkyleneimine-alkoxylene polymer or in absence of an alkoxylene unit.
  • a primary amine of PEI or polypropyleneimine may be functionalized with 0, 1 or more alkoxylene unit(s) (e.g., a hydrogen atom of the primary amine may be substituted with an alkoxylene unit).
  • Functionalizing one or more primary amines of PEI or polypropyleneimine with at least one alkoxylene unit may form a polyalkyleneimine- alkoxylene polymer of the present invention.
  • a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 1, 2, or 3 to about 4, 5, 6, or 8 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 8, 9, or 10 to about 11, 12, or 13 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 16, 17, or 18 to about 19, 20, 21, or 22 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer.
  • At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the primary amines of PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer are substituted/functionalized with at least one alkoxylene unit (e.g., at least one ethylene oxide unit).
  • the amount or degree of alkoxylation for a polyalkyleneimine-alkoxylene polymer may be measured using methods known to those of skill in the art such as by determining OH-number and/or by using nuclear magnetic resonance (e.g., 'H NMR).
  • alkoxylation may be quantified by determining the OH-number (also referred to interchangeably as an OH-value) for a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer), and an alkoxylation degree of 3-5 units may be about 320-213 mg KOH, respectively, to provide an OH-number of about 213 to about 320, an alkoxylation degree of 8-13 alkoxylation may be about 142-91 mg KOH, respectively, to provide an OH-number of about 91 to about 142, and/or an alkoxylation degree of 18-22 may be about 67-55 mg KOH, respectively, to provide an OH-number of about 55-66.
  • an alkoxylation degree of 3-5 units may be about 320-213 mg KOH, respectively, to provide an OH-number of about 213 to about 320
  • an alkoxylation degree of 8-13 alkoxylation may be about 142-91 mg KOH, respectively
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) has an OH-number in a range of about 40 to about 400, about 40 to about 100, about 100 to about 200, or about 200 to about 400.
  • a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine- alkoxylene polymer may be capped with hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl.
  • a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine-alkoxylene polymer is capped with hydrogen or a -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl.
  • a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is capped with hydrogen.
  • an internal and/or terminal nitrogen atom e.g., of a primary, secondary, and/or tertiary amine
  • PEI or polypropyleneimine of the polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • one or more nitrogen atom(s) in a polyalkyleneimine- alkoxylene polymer may be substituted with a substituent comprising 1 to 12 carbon atoms (also referred to herein as a C1-C12 substituent).
  • a C1-C12 substituent may be saturated or unsaturated and/or linear or branched.
  • a C1-C12 substituent is a saturated or unsaturated, linear or branched hydrocarbon.
  • a C1-C12 substituent is a substituted or unsubstituted C1-C12 alkyl, alkenyl, alkynyl, aryl, arylalkyl arylalkenyl, or arylalkynyl substituent.
  • one or more nitrogen atom(s) in a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • one or more nitrogen atom(s) in a polyalkyleneimine-alkoxylene polymer may be substituted with a benzyl.
  • one or more nitrogen atom(s) in a polyalkyleneimine- alkoxylene polymer may be substituted with a group selected from a C1-C4 alkyl, a C1-C4 aryl, and/or a C1-C4 arylalkyl.
  • Substitution with a C1-C12 substituent may result in a neutral or cationic charge on a respective nitrogen atom depending on its total number of substituents. In some embodiments, substitution with a C1-C12 substituent may result in permanent quaternization (i.e., a permanent cationic charge) at the substituted position. In some embodiments, substitution with a C1-C12 substituent may result in permanent quatemization of a nitrogen atom in the PEI or polypropyleneimine backbone of a polymer.
  • a polyalkyleneimine-alkoxylene polymer may comprise one or more (e.g., 1, 2, 3, 4, 5, 10, 20, 100, or more) quatemized functional group(s).
  • a nitrogen atom e.g., of a primary, secondary, and/or tertiary amine
  • the polymer may comprise one or more quaternized functional group(s).
  • about 0%, 25%, or 50% to about 60%, 70%, 80%, 90%, or 100% of the nitrogen atoms of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quatemized functional group.
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • about 0%, 5%, 10%, 20%, 40%, or 50% to about 60%, 70%, 80%, 90%, or 100% of the nitrogen atoms in the PEI or polypropyleneimine backbone of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quaternized functional group.
  • a quaternized functional group may comprise a nitrogen atom of PEI or polypropyleneimine bound to a substituent comprising 1 to 12 carbon atoms (also referred to herein as a C1-C12 substituent).
  • exemplary C1-C12 substituents include, but are not limited to, benzyl, a C1-C12 alkyl, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 arylalkyl, a C1-C12 arylalkenyl, or a C1-C12 arylalkynyl, each of which may be substituted or unsubstituted.
  • a quatemized functional group is formed by reacting a C1-C12 substituent with a tertiary amine of PEI or polypropyleneimine.
  • the polyalkyleneimine (e.g., PEI) and/or polyalkoxylene (e.g., PEO) of the polyalkyleneimine-alkoxylene polymer may be linear and/or branched. In some embodiments, PEI and/or PEO of a PEI-PEO polymer are branched.
  • a polyalkyleneimine-alkoxylene polymer may comprise 1, 5, 10, or 20 to 30, 40, or 50 alkoxylene units.
  • PEO of a PEI-PEO polymer may comprise 1, 5, 10, or 20 to 30, 40, or 50 ethylene oxide units.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises a polyalkoxylene in an amount of about 50% to about 99% by weight of the polymer such as about 75% to about 99%, about 80% to about 99%, about 80% to about 95%, or about 90% to about 95% by weight of the polymer.
  • a polyalkyleneimine-alkoxylene polymer may comprise a polyalkoxylene in an amount of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
  • a polyalkyleneimine-alkoxylene polymer comprises PEI or polypropyleneimine in an amount of about 0.5% to about 50% by weight of the polymer such as about 1% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 0.5% to about 10% by weight of the polymer.
  • a polyalkyleneimine-alkoxylene polymer comprises PEI or polypropyleneimine in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
  • a PEI-PEO polymer comprises PEO in an amount of about 50% to about 99% by weight of the PEI-PEO polymer such as about 75% to about 99%, about 80% to about 99%, about 80% to about 95%, or about 90% to about 95% by weight of the PEI-PEO polymer.
  • a PEI-PEO polymer may comprise PEO in an amount of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
  • PEI-PEO polymer comprises PEI in an amount of about 0.5% to about 50% by weight of the PEI-PEO polymer such as about 1% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 0.5% to about 10% by weight of the PEI-PEO polymer.
  • a PEI-PEO polymer comprises PEI in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% by weight of the PEI-PEO polymer.
  • PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer of the present invention may have an amine value in a range of about 10 or 15 to about 20, 25 or 30.
  • PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an amine value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, or 25.
  • amine value is the number of milligrams of potassium hydroxide (KOH) equivalent to the basicity in 1 gram of sample (e.g., 1 gram of polymer). Amine value may be measured in accordance with ASTM D 2073- 92 and optionally calculating the total amine value assuming all amines are primary amines.
  • KOH potassium hydroxide
  • PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has a molecular weight of about 400 g/mol to about 900,000 g/mol such as about 400 g/mol to about 825,000 g/mol, about 400 g/mol to about 750,000 g/mol, about 400 g/mol to about 10,000 g/mol, about 400 g/mol to about 600 g/mol, about 800 g/mol to about 20,000 g/mol, about 600,000 g/mol to about 900,000 g/mol, about 600,000 g/mol to about 825,000 g/mol, about 4,000 g/mol to about 6,000 g/mol, about 1,000 g/mol to about 500,000 g/mol, about 10,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 30,000 g/mol, or about
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an average molecular weight of about 1,000 g/mol to about 30,000,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 160,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has a molecular weight of about 1,000 g/mol to about 10,000,000 g/mol such as about 1,000 g/mol to about 10,000 g/mol, about 100,000 g/mol to about 1,000,000 g/mol, about 1,000,000 g/mol to about 10,000,000 g/mol, about 1,900 g/mol to about 2,900 g/mol, about 2,100 g/mol to about 2,400 g/mol, about 3,300 g/mol to about 5,000 g/mol, about 3,700 g/mol to about 4,600 g/mol, about 6,100 g/mol to about 9,300 g/mol, about 6,900 g/mol to about 8,500 g/mol, about 100,000 g/mol to about 160,000 g/mol, about 115,000 g/mol to about 141,000 g/mol, about 266,000 g/mol to about 400,000 g/mol, about 299,000
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an EO value (degree of ethoxylation) of about 2 to about 6, about 2 to about 20, about 2 to about 28, about 2 to about 30, about 3 to about 5, about 3 to about 6, about 3 to about 9, about 3 to about 15, about 3 to about 22, about 5 to about 28, about 7 to about 15, about 8 to about 14, or about 17 to about 30 or any range or value therein.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an OH-number of about 70 mg to about 40 mg, about 70 mg to about 50 mg, about 100 mg to about 40 mg, about 100 mg to about 60 mg, about 130 mg to about 40 mg, about 130 mg to about 80 mg, about 200 mg to about 40 mg, about
  • 300 mg to about 40 mg about 300 mg to about 50 mg, about 300 mg to about 60 mg, about 300 mg to about 70 mg, about 300 mg to about 80 mg, about 340 mg to about 60 mg, about 340 mg to about 80 mg, about 350 mg to about 40 mg, about 350 mg to about 50 mg, about 350 mg to about 70 mg, or about 350 mg to about 80 mg, or any range or value therein.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 20,000 g/mol to about 200,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, 20,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 90,000 g/mol, about 25,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 90,000 g/mol, about 30,000 g/mol to about 100,000 g/mol, about
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer had/has an amine value of about 15 to about 20 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 3,000,000 g/mol to about 30,000,000 g/mol, about 4,000,000 g/mol to about 12,000,000 g/mol, about 4,000,000 g/mol to about 15,000,000 g/mol, about 4,000,000 g/mol to about 20,000,000 g/mol, about 4,000,000 g/mol to about 25,000,000 g/mol, about 4,5000,000 g/mol to about 10,000,000 g/mol, about 4,5000,000 g/mol to about 12,000,000 g/mol, about 4,5000,000 g/mol to about 15,000,000 g/mol, about 4,5000,000 g/mol to about 20,000,000 g/mol, about 4,5000,000 g/mol to about 24,000,000 g/mol, about 10,000,000 g/mol to about 24,000,000 g/mol, about 15,000,000 g/mol to about 24,000,000 g/mol, about 20,000,000 g/mol,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 15 to about 20 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 120,000 g/mol to about 1,000,000 g/mol, about 145,000 g/mol to about 450,000 g/mol, about 145,000 g/mol to about 500,000 g/mol, about 145,000 g/mol to about 600,000 g/mol, about 145,000 g/mol to about 800,000 g/mol, about 150,000 g/mol to about 400,000 g/mol, about 150,000 g/mol to about 450,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 700,000 g/mol, about 150,000 g/mol to about 800,000 g/mol, about 350,000 g/mol to about 800,000 g/mol, about 400,000 g/mol to about 800,000 g/mol, about 121,000 g/mol to
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 15 to about 20 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 20,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 160,000 g/mol, about 25,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 140,000 g/mol, about 25,000 g/mol to about 150,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 25,000 g/mol to about 180,000 g/mol, about 25,000 g/mol to about 200,000 g/mol, about 25,000 g/mol to about 500,000 g/mol, about 25,000 g/mol to about 1,500,000 g/mol, about 25,000 g/mol to about 3,000,000 g/mol, about 25,000 g/mol to about 5,000,000 g/mol, about 25,000 g
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 75,000 g/mol to about 400,000 g/mol, about 75,000 g/mol to about 500,000 g/mol, about 75,000 g/mol to about 1,000,000 g/mol, about 75,000 g/mol to about 5,000,000 g/mol, about 75,000 g/mol to about 8,000,000 g/mol, about 75,000 g/mol to about 12,000,000 g/mol, about 80,000 g/mol to about 450,000 g/mol, about 80,000 g/mol to about 500,000 g/mol, about 80,000 g/mol to about 750,000 g/mol, about 80,000 g/mol to about 1,000,000 g/mol, about 80,000 g/mol to about 5,000,000 g/mol, about 100,000 g/mol to about 450,000 g/mol, about 100,000 g/mol to about 500,000 g/mol, about 100,000 g
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 150,000 g/mol to about 250,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 800,000 g/mol, bout 150,000 g/mol to about 900,000 g/mol, bout 150,000 g/mol to about 1,000,000 g/mol, about 150,000 g/mol to about 5,000,000 g/mol, about 150,000 g/mol to about 12,000,000 g/mol, 150,000 g/mol to about 24,000,000 g/mol, about 150,000 g/mol to about 25,000,000 g/mol, about 500,000 g/mol to about 1,000,000 g/mol, about 500,000 g/mol to about 5,000,000 g/mol, about 500,000 g/mol to about 10,000,000 g/mol, about 500,000 g/mol to about 12,000,000
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 25,000 g/mol to about 40,000 g/mol, about 25,000 g/mol to about 35,000 g/mol or any range or value therein, (e.g., about 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, or 40,000 g/mol), optionally, an average molecular weight of about 30,000 g/mol; a molecular weight of about 1,900 g/mol to about 2,900 g/mol or about 2,100 g/mol to about 2,400 g/mol; about 2 to about 5 alkoxylene units per amine of the polypropy
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 75,000 g/mol to about 90,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 75,000 g/mol to about 85,000 g/mol or any range or value therein, (e.g., about 75,000, 76,000, 77,000, 78,000, 79,000, 80,000, 81,000, 82,000, 83,000, 84,000, 85,000, 86,000, 87,000, 88,000, 89,000, or 90,000 g/mol), optionally, an average molecular weight of about 82,000 g/mol; a molecular weight of about 3,300 g/mol to about 5,000 g/mol or about 3,700 g/mol to about 4,600 g/mol; about 7 to about 10 alkoxylene units per amine of the poly
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 140,000 g/mol to about 180,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 140,000 g/mol to about 170,000 g/mol or any range or value therein, (e.g., about 140,000, 141,000, 142,000, 143,000, 144,000, 145,000, 146,000, 147,000, 148,000, 149,000, 150,000, 155,000, 156,000, 157,000, 158,000, 159,000, 160,000, 161,000, 162,000, 163,000, 164,000, 165,000, 166,000, 167,000, 168,000, 169,000, 170,000, 171,000, 172,000, 173,000, 174,000, 175,000, 176,000, 177,000, 178,000, 179,000 or 180,000 g/mol), optionally, an average molecular weight of about
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 4,000,000 g/mol to about 5,000,000 g/mol, about 3,600,000 g/mol to about 5,400,000 g/mol, about 4,000,000 g/mol to about 4,900,000 g/mol, or any range or value therein, (e.g., about 4,000,000,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 10,000,000 g/mol to about 15,000,000 g/mol, about 9,000,000 g/mol to about 15,000,000 g/mol, about 10,000,000 g/mol to about 13,000,000 g/mol, or any range or value therein, (e.g., about
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 22,000,000 g/mol to about 25,000,000 g/mol, about 19,000,000 g/mol to about 29,000,000 g/mol, about 17,000,000 g/mol to about 26,000,000 g/mol, or any range or value therein, (e.g., about
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 140,000 g/mol to about 180,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about 167,000 g/mol, about 140,000 g/mol to about 170,000 g/mol or any range or value therein, (e.g., about 140,000, 141,000, 142,000, 143,000, 144,000, 145,000, 146,000, 147,000, 148,000, 149,000, 150,000, 155,000, 156,000, 157,000, 158,000, 159,000, 160,000, 161,000, 162,000, 163,000, 164,000, 165,000, 166,000, 167,000, 168,000, 169,000, 170,000, 171,000, 172,000, 173,000, 174,000, 175,000, 176,000, 177,000, 178,000, 179,000 or
  • 180,000 g/mol optionally, an average molecular weight of about 152,000 g/mol; a molecular weight of about 100,000 g/mol to about 160,000 g/mol or about 115,000 g/mol to about 141,000 g/mol,; about 2 to about 6 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
  • alkoxylene e.g., ethylene oxide
  • a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 5 nm to about 15 nm such as about 10 nm
  • a zeta potential in a range of about +4 optionally when present in a composition having a pH of about 7; and/or an OH-number of about 240 mg to about 190 mg, about 220 mg to about 200 mg, or any range
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 300,000 g/mol to about 500,000 g/mol, about 326,000 g/mol to about 490,000 g/mol, about 367,000 g/mol to about 448,000 g/mol, about 350,000 g/mol to about 450,000 g/mol or any range or value therein, (e.g., about 300,000, 305,000, 310,000, 315,000, 320,000, 325,000, 340,000, 345,000, 350,000, 355,000, 360,000, 365,000, 370,000, 375,000, 380,000, 385,000, 390,000, 395,000, 400,000, 401,000, 402,000, 403,000, 404,000, 405,000, 406,000, 407,000, 408,000, 409,000, 410,000, 415,000, 420,000, 425,000, 430,000, 435,000, 440,000, 450,000, 460,000, 470,000, 480,000, 490,000,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 650,000 g/mol to about 850,000 g/mol, about 633,000 g/mol to about 950,000 g/mol, about 712,000 g/mol to about 871,000 g/mol, about 700,000 g/mol to about 800,000 g/mol or any range or value therein, (e.g., about 650,000, 660,000, 680,000, 690,000, 700,000, 710,000, 720,000, 730,000, 740,000, 750,000, 760,000, 780,000, 781,000, 782,000, 783,000, 784,000, 785,000, 786,000, 787,000, 788,000, 789,000, 790,000, 791,000, 792,000, 793,000, 794,000, 795,000, 796,000, 797,000, 798,000, 799,000, 800,000, 810,000,
  • an average molecular weight of about 792,000 g/mol optionally, an average molecular weight of about 792,000 g/mol; a molecular weight of about 520,000 g/mol to about 782,000 g/mol or about 585,000 g/mol to about 717,000 g/mol; about 25 to about 30 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a portion or all of the nitrogen atoms in PEI or polypropyleneimine of a polyalkyleneimine-alkoxylene polymer of the present invention may be substituted with at least one alkoxylene unit (e.g., at least one ethylene oxide unit).
  • a portion or all of the nitrogen atoms in PEI of a PEI-PEO polymer of the present invention may be substituted with at least one alkoxylene unit (e.g., at least one ethylene oxide unit).
  • Scheme 1 Exemplary alkoxylation modification for a terminal nitrogen atom in a polyalkyleneimine.
  • a further exemplary alkoxylation modification is shown in Scheme 2 for an internal nitrogen atom in the polyalkyleneimine backbone of a polyalkyleneimine-alkoxylene polymer where R represents an ethylene spacer and E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl) and X represents a water soluble counterion.
  • R represents an ethylene spacer
  • E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl)
  • X represents a water soluble counterion.
  • Scheme 2 Exemplary alkoxylation modification for an internal nitrogen atom in a polyalkyleneimine.
  • an alkoxylation modification of a polyalkyleneimine backbone may comprise substitution of a hydrogen atom with a polyalkoxylene chain having an average of about 1 to about 50 alkoxy units such as about 2 to about 40 alkoxy units, about 3 to about 30 units, or about 3 to about 20 alkoxy units.
  • Exemplary alkoxy units include, but are not limited to, ethoxy (EO), 1,2-propoxy (1,2-PO), and/or butoxy (BO).
  • a polyalkoxylene chain comprises, consists essentially of, or consists of ethoxy units.
  • a polyalkoxylene chain comprises, consists essentially of, or consists of ethoxy units and propoxy units.
  • a polyalkoxylene chain comprising, consisting essentially of, or consisting of ethoxy units and propoxy units may comprise, on average, about 1 to about 20 or 30 ethoxy units and, on average, about 0 or 1 to about 10 propoxy units.
  • An exemplary alkoxylated polyethyleneimine polymer of the present invention may have a structure of Formula (I):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl; and
  • each n is an integer of 1 to 50.
  • An exemplary quaternized alkoxylated polyethyleneimine polymer of the present invention may have a structure of formula (II):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
  • each E is independent a Cl -Cl 2 substituent
  • each X is independently a water soluble counterion (e.g., chlorine, bromine, iodine, sulphate, alkyl sulfonate, etc.); and
  • each n is an integer of 1 to 50.
  • a compound of Formula (I) and/or (II) may comprise a polyalkyleneimine (e.g., polyethyleneimine) backbone with a weight average molecular weight of about 600 g/mole to about 500,0000 g/mole.
  • n may be determined based on an average number of units such as, n, on average, may be an integer of 1 to 30 or 50.
  • the degree of quaternization of nitrogen atoms in the polyalkyleneimine backbone of Formula (II) may be at least 5%, 20%, 70% or more.
  • an exemplary alkoxylated polyethyleneimine polymer of the present invention may have a structure of Formula (III):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
  • each n is an integer of 0 to 50;
  • each m is an integer of 1 to 10.
  • structure of the polymer of formula (III) is not limited to block structures.
  • structure of the polymer of formula (III) comprises heteric blocks (e.g., blocks where EO and PO are randomly mixed).
  • Another exemplary quaternized alkoxylated polyethyleneimine polymer of the present invention may have a structure of formula (IV):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
  • each E is independent a Cl -Cl 2 substituent
  • each X is independently a water soluble counterion (e.g., chlorine, bromine, iodine, sulphate, alkyl sulfonate, etc.);
  • each n is an integer of 1 to 50;
  • each m is an integer of 1 to 10.
  • structure of the polymer of formula (IV) is not limited to block structures.
  • structure of the polymer of formula (IV) comprises heteric blocks (e.g., blocks where EO and PO are randomly mixed).
  • a compound of Formula (III) and/or (IV) may comprise a polyalkyleneimine (e.g., polyethyleneimine) backbone with a weight average molecular weight of about 600 g/mole to about 500,0000 g/mole.
  • n may be determined based on an average number of units such as, n, on average, may be an integer of 1 to 20, 30, or 50.
  • m may be determined based on an average number of units such as, m, on average, may be an integer of 1 to 10.
  • the degree of quatemization of nitrogen atoms in the polyalkyleneimine backbone of Formula (IV) may be at least 5%, 20%, 70% or more.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may be prepared in a known manner by reaction of polyalkyleneimines with alkoxy units, an exemplary process is described for the ethoxylation of polyethyleneimine.
  • a method of preparing a polyalkyleneimine-alkoxylene polymer of the present invention comprises, consists essentially of, or consists of alkoxylation (e.g., ethoxylation) of a polyalkyleneimine (e.g., PEI).
  • the polyalkyleneimine may be reacted with all or a portion of the total amount of an alkylene oxide (e.g., ethylene oxide) used in the alkoxylation reaction.
  • a portion or a total amount of alkylene oxide used in the alkoxylation reaction may be about 1 mole of alkylene oxide (e.g., ethylene oxide) per amine (based on total amine value) of polyalkyleneimine.
  • An alkoxylation reaction may be undertaken in the absence of a catalyst and/or in an aqueous solution at a reaction temperature in a range of about 70°C to about 200°C.
  • the alkoxylation reaction is carried out in the presence of water at a reaction temperature in a range of about 80°C to about 160°C and in the absence of a catalyst. In some embodiments, the alkoxylation reaction is carried out at a pressure of up to about 5, 6, 7, 8, 9, or 10 bar.
  • a polyalkyleneimine e.g., PEI
  • a first portion of alkylene oxide e.g., ethylene oxide
  • an at least partially alkoxylated polyalkyleneimine e.g., at least partially alkoxylated PEI
  • the at least partially alkoxylated polyalkyleneimine is reacted with a second portion of an alkylene oxide.
  • PEI polyalkyleneimine
  • ethylene oxide as the exemplary alkylene oxide
  • the at least partially ethoxylated PEI is reacted with a second portion of an ethylene oxide.
  • the second portion of ethylene oxide is the final ethylene oxide addition and/or the second portion in combination with the first portion of ethylene oxide (and/or any other portions) makes up the total amount of ethylene oxide used in the ethoxylation reaction.
  • reaction of the at least partially ethoxylated PEI and the second portion of ethylene oxide is carried out in the presence of a basic catalyst.
  • Examples of basic catalysts include, but are not limited to, alkali metal hydroxides and alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide and/or calcium hydroxide; alkali metal alkoxides such as sodium and potassium Ci-C4-alkoxides (e.g., sodium methoxide, sodium ethoxide and potassium tert-butoxide); alkali metal and alkaline earth metal hydrides such as sodium hydride and calcium hydride; and/or alkali metal carbonates such as sodium carbonate and potassium carbonate.
  • the basic catalyst is an alkali metal hydroxide or alkali metal alkoxide such as potassium hydroxide and/or sodium hydroxide.
  • a basic catalyst may be used and/or present in an amount of about 0.05% to about 10% by weight of polyalkyleneimine and the alkylene oxide.
  • the at least partially alkoxylated polyalkyleneimine e.g., at least partially ethoxylated PEI
  • a second portion of alkylene oxide e.g., ethylene oxide
  • reacting an at least partially alkoxylated polyalkyleneimine e.g., at least partially ethoxylated PEI
  • a second portion of alkylene oxide e.g., ethylene oxide
  • reacting an at least partially alkoxylated polyalkyleneimine e.g., at least partially ethoxylated PEI
  • a second portion of alkylene oxide e.g., ethylene oxide
  • the method will be further described with PEI as the exemplary polyalkyleneimine and ethylene oxide as the exemplary alkylene oxide.
  • the at least partially ethoxylated PEI may be dewatered, optionally in the presence of a basic catalyst.
  • Dewatering may be carried out by heating a composition comprising the at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) and optionally basic catalyst to a temperature of about 80°C or 120°C to about 150°C or 180°C and distilling off the water under a reduced pressure of about 0.01 bar to about 0.5 bar.
  • the dewatering may be carried out and/or supported by a gentle nitrogen stream.
  • a subsequent reaction of the at least partially ethoxylated PEI with a second portion of ethylene oxide in a dewatered composition in the presence of a basic catalyst may be effected at a reaction temperature from about 70°C to about 200°C or about 100°C to about 180°C and/or at a pressure of up to about 8 or 10 bar.
  • reacting an at least partially ethoxylated PEI with a second portion of ethylene oxide in a dewatered composition and in the presence of a basic catalyst is carried out for a period of time of about 30 minutes to about 4 hours.
  • the PEI-PEO polymer may be obtained directly in substance or may be obtained in and/or converted to an aqueous solution.
  • reacting an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) with a second portion of alkylene oxide (e.g., ethylene oxide) is carried out in an organic solvent such as a nonpolar organic solvent and/or polar aprotic organic solvent.
  • organic solvent such as a nonpolar organic solvent and/or polar aprotic organic solvent.
  • nonpolar aprotic solvents include, but are not limited to, aliphatic and aromatic hydrocarbons such as hexane, cyclohexane, toluene and xylene.
  • Exemplary polar aprotic solvents include, but are not limited to, ethers such as cyclic ethers (e.g., tetrahydrofuran and dioxane), N,N-dialkylamides such as dimethylformamide and dimethylacetamide, and/or N-alkyl lactams such as N-methylpyrrolidone.
  • the organic solvent comprises two or more (e.g., 2, 3, 4 or more) organic solvents.
  • the organic solvent comprises xylene and/or toluene.
  • a composition comprising an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI), a basic catalyst, and an organic solvent may be dewatered as described herein to provide a dewatered composition.
  • dewatering is carried out by separating out the water from the composition at a temperature of about 120°C to about 180°C, optionally supported by a gentle nitrogen stream.
  • a subsequent reaction of the at least partially alkoxylated polyalkyleneimine with a second portion of alkylene oxide in an organic solvent in the presence of a basic catalyst may be effected at a reaction temperature from about 70°C to about 200°C or about 100°C to about 180°C and/or at a pressure of up to about 8 or 10 bar.
  • the obtained polyalkyleneimine- alkoxylene polymer e.g., PEI-PEO polymer
  • the organic solvent may be removed and optionally replaced with water.
  • the polyalkyleneimine-alkoxylene polymer e.g., PEI-PEO polymer
  • Quaternization may be carried out with methods known to those of skill in the art.
  • quaternization of a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • quaternization of a polyalkyleneimine-alkoxylene polymer is achieved by introducing a Cl- C12 alkyl, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 arylalkyl, a Cl- C12 arylalkenyl, or a Cl -Cl 2 arylalkynyl group such as by reaction of the polymer with the corresponding C1-C12 alkyl- halide or dialkylsulfate, C1-C12 alkenyl- halide or dialkylsulfate, C1-C12 alkynyl- halide or dialkylsulfate, C
  • quaternization of a polyalkyleneimine-alkoxylene polymer is achieved by reacting an amine of the polymer with at least one alkylating compound.
  • the at least one alkylating compound may be represented by the chemical formula EX, wherein E is a C1-C12 alkyl, a C1-C12 alkenyl, a Cl -Cl 2 alkynyl, a Cl -Cl 2 aryl, a Cl -Cl 2 arylalkyl, a Cl -Cl 2 arylalkenyl, or a Cl -Cl 2 arylalkynyl and X is a leaving group that is capable of being replaced by nitrogen or a C2-C6 alkylene oxide (e.g., ethylene oxide or propylene oxide).
  • E is a C1-C12 alkyl, a C1-C12 alkenyl, a Cl -Cl 2 alkynyl, a Cl -Cl 2 aryl, a Cl -Cl 2 arylalkyl, a Cl -Cl 2 arylalkenyl, or a Cl -Cl
  • Exemplary leaving groups include, but are not limited to, halogens (e.g., chlorine, bromine, or iodine), sulphate, alkyl sulfonate (e.g., methyl sulfonate), arylsulfonate (e.g., tolyl sulfonate), and alkyl sulphate (e.g., methosulphate).
  • halogens e.g., chlorine, bromine, or iodine
  • alkyl sulfonate e.g., methyl sulfonate
  • arylsulfonate e.g., tolyl sulfonate
  • alkyl sulphate e.g., methosulphate
  • Exemplary alkylating agents include, but are not limited to, Cl -Cl 2 alkyl halides, bis(Cl-C12-alkyl)sulfates, and benzyl hal
  • the at least one alkylating agent is selected from ethyl chloride, ethyl bromide, methyl chloride, methyl bromide, benzyl chloride, dimethyl sulphate, and/or diethyl sulphate.
  • the method may comprise combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer), thereby preparing the composition.
  • the composition may comprise two or more polyalkyleneimine-alkoxylene polymers and/or two or more nucleic acid molecules.
  • the polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • is complexed with the nucleic acid molecule optionally wherein a single polymer is complexed with 2, 5, 10, 100, or more nucleic acid molecules as described herein.
  • a single nucleic acid molecule is complexed with 2, 5, 10, 25, 50, 75, 100, or more polyalkyleneimine-alkoxylene polymers as described herein.
  • Combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer may be carried out by contacting a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer either of which may be present in the form of a solution or a solid.
  • the nucleic acid molecule and/or the polyalkyleneimine-alkoxylene polymer is/are present in the form of a solid that is added to a solution (e.g., an aqueous solution).
  • combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer comprises mixing a first solution comprising the nucleic acid molecule and a second solution comprising the polymer to form a third solution comprising the nucleic acid molecule and the polymer.
  • combining a nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer comprises providing the nucleic acid molecule and the polymer in contact for a period of time such as about 1, 5, 10, 15, or 30 minutes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.
  • combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer comprises providing the nucleic acid molecule and the polymer in the same composition (e.g., an aqueous solution) for a period of time, optionally with mixing and/or agitation.
  • the polymer may be dialyzed prior to combining/contact with the nucleic acid (e.g., the polymer may be dialyzed about 1, 5, 10, 15, 20, 30, 40, 50 minutes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours prior to contact with the nucleic acid.
  • compositions comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) that can be used to deliver the nucleic acid molecule to a cell (e.g. host cell) of an organism (e.g., a target organism).
  • a cell e.g. host cell
  • an organism e.g., a target organism.
  • the organism from which a cell may be obtained can be any eukaryote (e.g., animal, plant, fungal, protozoa, and the like).
  • An animal useful with this invention may be any animal including but not limited to, a mammal, an insect, a plant, a fungus, a bird, a fish, an amphibian, a reptile, or a cnidarian.
  • a mammal can include, but is not limited to, a rodent, a horse, a dog a cat, a human, a non human primate (e.g., monkeys, baboons, and chimpanzees), a goat, a pig, a cow (e.g., cattle), a sheep, laboratory animals (e.g, rats, mice, gerbils, hamsters, and the like) and the like.
  • compositions of the invention comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) can be used to deliver the nucleic acid molecule to, for example, a cell of a cell line (e.g., a mammalian cell line, an insect cell line).
  • a cell of a cell line e.g., a mammalian cell line, an insect cell line.
  • mammalian and insect cell lines include HEK293 cells, HeLa cells, CHO cells, MEF cells, 3T3 cells, Hi-5 cells, and Sf21 cells.
  • Suitable target organisms can include both males and females and subjects of all ages including embryonic (e.g, in utero or in ovo), infant, juvenile, adolescent, adult and geriatric subjects.
  • the target organism is not a human subject or a human embryonic subject.
  • a plant, plant part, and/or plant cell useful with this invention can include, but not is not limited to, Camelina, Glycine, Sorghum, Brassica, Allium, Armoracia, Poa , Solanum, Cucurbita, Musa, Agrostis, Lolium, Festuca, Calamogrostis, Deschampsia, Spinacia, Beta, Pisum, Chenopodium, Helianthus, Pastinaca, Daucus, Petroselium, Populus, Prunus, Castanea, Eucalyptus, Acer, Quercus, Salix, Juglans, Picea, Pinus, Mains, Abies , Lemna , Wolffia , Spirodela, Oryza, Zea or Gossypium.
  • the plant and/or plant cell can include, but is not limited to, Camelina alyssum (Mill.) Thell., Camelina microcarpa Andrz. ex DC., Camelina rumelica Velen., Camelina sativa (L.) Crantz, Sorghum bicolor (e.g., Sorghum bicolor L.
  • a plant and/or plant cell can be, but is not limited to, wheat, barley, rye, com, oats, turfgrass (bluegrass, bentgrass, ryegrass, fescue), sugar cane, feather reed grass, tufted hair grass, spinach, cucumber, melon, leak, tomato, potato, beets, chard, quinoa, sugar beets, lettuce, sunflower ( Helianthus annuus ), peas (Pisum sativum), parsnips ( Pastinaca sativa), carrots ( Daucus carota), parsley (Petroselinum crispum), duckweed, pine, spruce, fir, eucalyptus, oak, walnut, or willow.
  • the plant and/or plant cell can be Arabidopsis thaliana.
  • the plant and/or plant cell can be camelina, wheat, rice, com, rape, canola, soybean, sorghum, tomato, bamboo or cotton.
  • plant part includes reproductive tissues (e.g, petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g, petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g, phloem and xylem); specialized cells such as epidermal cells, parenchyma cells,
  • plant part also includes plant cells, including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like.
  • plant protoplasts plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like.
  • shoot refers to the above ground parts including the leaves and stems.
  • tissue culture encompasses cultures of tissue, cells, protoplasts and callus.
  • plant cell refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts.
  • a plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ.
  • a plant cell can be an algal cell.
  • the polymers of this invention will be especially useful in transforming intact plant cells (e.g., cells with cell walls, e.g., plants and plant tissues), providing a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium and the like.
  • intact plant cells e.g., cells with cell walls, e.g., plants and plant tissues
  • a plant, plant part, and/or plant cell can be an algae or algae cell including, but not limited to, a Bacillariophyceae (diatoms), Haptophyceae, Phaeophyceae (brown algae), Rhodophyceae (red algae) or Glaucophyceae (red algae).
  • a Bacillariophyceae diatoms
  • Haptophyceae Haptophyceae
  • Phaeophyceae brown algae
  • Rhodophyceae red algae
  • Glaucophyceae red algae
  • non-limiting examples of an algae or algae cell can be Achnanthidium , Actinella, Nitzschia, Nupela, Geissleria, Gomphonema, Planothidium, Halamphora, Psammothidium, Navicula, Eunotia, Stauroneis, Chlamydomonas, Dunaliella, Nannochloris, Nannochloropsis, Scenedesmus, Chlorella, Cyclotella, Amphora, Thalassiosira , Phaeodactylum, Chrysochromulina, Prymnesium, Thalassiosira, Phaeodactylum, Glaucocystis, Cyanophora, Galdieria, or Porphyridium.
  • Non-limiting examples of fungi useful with this invention include Candida spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Tinea spp., Sporothrix spp., Blastomyces spp., Histoplasma spp., Pneumocystis spp, Saccharomyces spp., Saccharomycodes spp., Hansenula spp., Kluyveromyces spp., Yarrowia spp., Pichia spp., Candida spp., Ashbya spp., Zygosaccharomyces spp.
  • the fungus can include, but is not limited to, Saccharomyces cerevisiae, S. uvarum (carlsbergensis), S. diastaticus, Saccharomycodes ludwigii, Hansenula polymorpha, Kluveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia pastor is, Pichia methanolica, Candida stellata, C.
  • a fungal cell useful with the invention may include its cell wall or may be without a cell wall, e.g., a spheroplast.
  • the polymers of the invention may be used to transfer nucleic acids to organelles, including, but not limited to, chloroplasts or mitochondria.
  • an organelle to be transformed as described herein may be isolated from the natural cellular environment.
  • One or more cells of an organism may be transformed with one or more nucleic acid molecules according to embodiments of the present invention.
  • one or more nucleic acid molecules may be delivered to a host cell or to one or more host cells in a population.
  • the cells in a population may be identical or different (e.g., cells having a different genetic background).
  • a population may comprise cells of at least two different species and/or strains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different species and/or strains).
  • a cell useful with this invention may be in growth phase (e.g., a yeast cell or population in growth phase). In some embodiments, a cell useful with this invention may be in stationary phase (e.g., a yeast cell or population in stationary phase.
  • compositions of the invention comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid as described herein are able to transform eukaryotic cells (e.g., generate transformants that have received DNA) and overcome typical problem(s) associated with other transformation protocols
  • a method of delivering a nucleic acid molecule to a host cell may comprise contacting the cell with a composition of the present invention, thereby delivering/introducing the nucleic acid molecule in the composition to/into the cell and/or transforming the cell with the nucleic acid molecule.
  • the composition may be in the form of a solid (e.g., that may be added to a solution comprising a cell to be transformed) or it may be a solution (e.g., an aqueous solution).
  • the composition is and/or comprises a complex comprising the nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer), optionally in an aqueous solution.
  • a polyalkyleneimine- alkoxylene polymer e.g., a PEI-PEO polymer
  • compositions of this invention i.e., a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and a nucleic acid molecule
  • a composition comprising a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • nucleic acid molecule e.g., polyalkyleneimine-alkoxylene polymer/nucleic acid molecule
  • “contact” or“contacting” can include contacting target nucleic acid with a composition of this invention in a cell free system, thereby delivering the nucleic acid molecule to the target nucleic acid (for example for modifying the target nucleic acid).
  • nucleic acid molecules may be assembled as part of a single nucleic acid molecule or nucleic acid construct, or as separate nucleic acid molecules or nucleic acid constructs, and can be located on the same or different expression constructs or transformation vectors.
  • a construct and/or vector may be present in a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the invention and/or complexed with the same or different polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers).
  • one or more polynucleotide(s) can be introduced into cells in a single transformation/transfection event, in separate transformation/transfection events, or any combination thereof.
  • one or more nucleic acid molecules of this invention can be introduced singly or in combination into a cell of a host organism.
  • “contacting” or “introducing” means contacting the population with a composition of the invention under conditions where at least the nucleic acid molecule gains access to the interior of one or more cells of the population, thereby transforming the one or more cells (e.g., stably or transiently).
  • contacting or “introducing” means contacting the population with a composition of the invention under conditions where the composition (e.g., polyalkyleneimine-alkoxylene polymer/ nucleic acid molecules) gains access to the interior of one or more cells of the population, thereby transforming the one or more cells of the population (e.g., stably or transiently).
  • a composition of the invention e.g., polyalkyleneimine-alkoxylene polymer/ nucleic acid molecules
  • “contacting” or“introducing” a composition of the invention i.e., a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule
  • a cell may be contacted and/or incubated with a composition of the present invention for about 0.5, 1, 2,
  • 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, or 24 hours, and any value or range therein) e.g., about 5 sec to about 30 min, about 1 min to about 1 hour, about 30 min to about 3 hours, about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 15 hours, about 1 hour to about 20 hours, about 1 hour to about 24 hours, about 5 hours to 10 hours, about 5 hours to 20 hours, about 10 hours to about 20 hours, about 10 hours to about 24 hours, or longer, or any value or range therein).
  • the incubation time may vary so long as the time is sufficient for the composition (i.e., the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule) to gain access to the interior of the cell.
  • the composition i.e., the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule
  • a cell may be contacted and/or incubated with a composition of the present invention at a pH from about 0 to about 14 (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 and any value or range therein).
  • a cell may be contacted and/or incubated with a composition of the present invention at a pH from about 3 to about 10.
  • a composition including a cell, nucleic acid molecule, and polyalkyleneimine-alkoxylene polymer may have a pH of about 6, 7, or 8 to about 8.5, 9, or 9.5.
  • a composition including a cell, nucleic acid molecule, and polyalkyleneimine- alkoxylene polymer has a pH of about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.
  • a cell may be contacted and/or incubated with a composition of the present invention at a temperature from about 0°C to about 100°C (e.g., about 1, 2, 3,
  • a cell may be contacted and/or incubated with a composition of the present invention at a temperature from about 4°C to about 50°C or about 15°C to about 37°C.
  • a method of the present invention delivers a nucleic acid molecule to a cell and/or a population of cells without adversely affecting the viability of the cell and/or population.
  • growth of such cells may be delayed or even prevented at an amount above about 5% weight/vol. of the polymer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% wt/vol. of the polymer.
  • a nucleic acid molecule of the invention may be operatively associated with a variety of promoters and other regulatory elements for expression in host cells.
  • a recombinant nucleic acid molecule of this invention may further comprise one or more promoters operably linked to one or more nucleotide sequences.
  • By“operably linked” or“operably associated” as used herein it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • the term“operably linked” or“operably associated” as used herein refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence.
  • control sequences e.g, promoter
  • the control sequences need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered“operably linked” to the nucleotide sequence.
  • A“promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription.
  • promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence.
  • the promoter region may comprise other elements that act as regulators of gene expression.
  • CAAT box consensus sequence examples include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349).
  • the CAAT box may be substituted by the AGGA box (Messing et al ., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).
  • Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e.,“chimeric genes” or“chimeric polynucleotides.” These various types of promoters are known in the art.
  • promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.
  • promoters include, but are not limited to, promoters functional in eukaryotes including but not limited to, plants, fungi, animals (e.g., mammals, insects, fish, amphibians, reptiles and the like).
  • Exemplar ⁇ promoters useful with yeast can include a promoter from phosphoglycerate kinase ( PGK ), glyceraldehyde-3 -phosphate dehydrogenase (GAP), triose phosphate isomerase (777), galactose-regulon ( GAL1 , GAL10 ), alcohol dehydrogenase (ADH1, ADH2), phosphatase (77/05), copper-activated metallothionine ( CUP1 ), MFal, PGK/a2 operator, TPI/a2 operator, GAP/GAL, PGK/GAL, GAP/ADH2, GAP/PH05, iso-1- cytochrome c/glucocorticoid response element (CYC/GRE), phosphoglycerate kinase/angrogen response element ( PGK/ARE), transcription elongation factor EF-la ( TEF1 ), triose phosphate dehydrogenase ( T
  • Non-limiting examples of a promoter functional in a plant include the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154 (2010)). PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters.
  • PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters.
  • Pnr is induced by nitrate and repressed by ammonium (Li et al. Gene 403: 132-142 (2007)) and Pdcal is induced by salt (Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)).
  • constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (U.S. Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as US Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad.
  • Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632), and arabidopsis (Norris et al. 1993. Plant Molec. Biol. 21 :895-906).
  • the maize ubiquitin promoter ( UbiP ) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926.
  • the ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons.
  • the promoter expression cassettes described by McElroy et al. can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledonous hosts.
  • tissue specific/tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell.
  • tissue- specific promoters include those associated with genes encoding the seed storage proteins (such as b-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g. , Kridl et al. (1991) Seed Sci. Res.
  • tissue- specific/tissue preferred promoters include, but are not limited to, the root hair-specific cis- elements (RHEs) (Kim et al. The Plant Cell 18:2958-2970 (2006)), the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153: 185-197 (2010)) and RB7 (U.S. Patent No. 5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11 : 160-167; and Vodkin (1983) Prog. Clin. Biol. Res.
  • RHEs root hair-specific cis- elements
  • promoters functional in chloroplasts can be used.
  • Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516.
  • Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).
  • inducible promoters can be used.
  • chemical -regulated promoters can be used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the RNAs and/or the polypeptides of the invention to be synthesized only when, for example, a crop of plants are treated with the inducing chemicals.
  • the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • a promoter can also include a light- inducible promoter, where application of specific wavelengths of light induce gene expression (Levskaya et al. 2005. Nature 438:441-442).
  • a promoter can include a light-repressible promoter, where application of specific wavelengths of light repress gene expression (Ye et al. 2011. Science 332: 1565-1568).
  • Chemical inducible promoters useful with plants are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid ( e.g ., the PRla system), steroid-responsive promoters (see, e.g, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al.
  • promoters useful with algae include, but are not limited to, the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep.
  • the promoter of the o 70 -type plastid rRNA gene (Prrn), the promoter of the psbA gene (encoding the photosystem -II reaction center protein Dl) (PpsbA), the promoter of the psbD gene (encoding the photosystem-II reaction center protein D2) (PpsbD), the promoter of the psaA gene (encoding an apoprotein of photosystem I) (PpsaA), the promoter of the ATPase alpha subunit gene (PatpA), and promoter of the RuBisCo large subunit gene (PrbcL), and any combination thereof (See, e.g., De Cosa et al.
  • a promoter useful with this invention can include, but is not limited to, pol III promoters such as the human U6 small nuclear promoter (U6) and the human HI promoter (HI) (Makinen et al. J Gene Med. 8(4):433-41 (2006)), and pol II promoters such as the CMV (Cytomegalovirus) promoter (Barrow et al. Methods in Mol. Biol.
  • pol III promoters such as the human U6 small nuclear promoter (U6) and the human HI promoter (HI) (Makinen et al. J Gene Med. 8(4):433-41 (2006)
  • pol II promoters such as the CMV (Cytomegalovirus) promoter (Barrow et al. Methods in Mol. Biol.
  • the SV40 Sema virus 40-derived initial promoter
  • the EF-la Elongation Factor-la
  • the Ubc Human Ubiquitin C
  • the PGK Mitine Phosphogly cerate Kinase- 1 promoter and/or constitutive protein gene promoters such as the b-actin gene promoter, the tRNA promoter and the like.
  • tissue-specific regulated nucleic acids and/or promoters as well as tumor- specific regulated nucleic acids and/or promoters have been reported.
  • tissue-specific or tumor-specific promoters can be used.
  • Some reported tissue- specific nucleic acids include, without limitation, B29 (B cells), CD 14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin (muscle), elastase-1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin (differentiating cells and healing tissues), FLT-1 (endothelial cells), GFAP (astrocytes), GPIIb (megakaryocytes), ICAM-2 (endothelial cells), INF-b (hematopoietic cells), Mb (muscle), NPHSI (podocytes), OG-2 (osteoblasts, SP-B (lungs),
  • tumor-specific nucleic acids and promoters include, without limitation, AFP (hepatocellular carcinoma), CCKAR (pancreatic cancer), CEA (epithelial cancer), c-erbB2 (breast and pancreatic cancer), COX-2, CXCR4, E2F-1, HE4, LP, MUC1 (carcinoma), PRC1 (breast cancer), PSA (prostate cancer), RRM2 (breast cancer), survivin, TRPl (melanoma), and TYR (melanoma).
  • inducible promoters can be used in mammalian cells.
  • inducible promoters include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper-inducible system promoters, salicylate- inducible system promoters ( e.g ., the PRla system), glucocorticoid-inducible promoters, and ecdysone-inducible system promoters.
  • a recombinant nucleic acid molecule of the invention can be an “expression cassette” or can be comprised within an expression cassette.
  • expression cassette means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleotide sequences of the invention; e.g., CRISPR nucleic acids), wherein said nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter).
  • a control sequence e.g., a promoter
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e ., termination region) that is functional in the selected host cell.
  • a transcriptional and/or translational termination region i.e ., termination region
  • a variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation.
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (, i.e ., foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
  • An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed host cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g ., an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., fluorescence).
  • a selective agent e.g ., an antibiotic and the like
  • screening e.g., fluorescence
  • vector refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell.
  • a vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced.
  • Vectors for use in transformation of host organisms are well known in the art.
  • Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • a vector as defined herein can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast, insect, fungi, and the like).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
  • the vector may be a bi functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • the nucleic acid molecules of this invention and/or expression cassettes may be comprised in vectors as described herein and as known in the art.
  • the terms“transformed” and“transgenic” refer to any cell that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide even if temporarily (i.e., transient) (e.g., CRISPR nucleic acid molecules)
  • transformation refers to the introduction of a nucleic acid molecule into a cell. Transformation of a cell may be stable or transient.
  • Transient transformation in the context of a nucleic acid molecule means that a nucleic acid molecule is introduced into the cell and does not integrate into the genome of the cell.
  • nucleic acid molecule introduced into a cell By stably introducing or stably introduced in the context of a nucleic acid molecule introduced into a cell it is intended that the introduced nucleic acid molecule is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the nucleic acid molecule.
  • “Stable transformation” or“stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Genome as used herein also includes the nuclear/chromosomal and the plasmid genome, and therefore includes integration of the nucleic acid molecule into, for example, the plasmid genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.
  • an“extrachromosomal nucleic acid” refers to select nucleic acids in eukaryotic cells such as in a mitochondrion, a plasmid, a plastid (e.g., chloroplast,
  • an extrachromosomal nucleic acid may be referred to as“extranuclear DNA” or“cytoplasmic DNA”
  • Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism.
  • Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant).
  • Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism.
  • Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols that are well known in the art.
  • PCR polymerase chain reaction
  • Transcriptional control means modulating expression of a target nucleic acid (e.g., target DNA) (i.e., activation (increasing) and/or repression (decreasing) expression of the target DNA or target RNA).
  • a deactivated Cas9 polypeptide can be fused with a transcriptional activator, thereby activating transcription of a target nucleic acid, thereby increasing expression of said target nucleic acid.
  • Any transcriptional activator now known or later identified can be used with this invention in a fusion construct with a Cas9 polypeptide as described herein.
  • a non-limiting example of such a transcriptional activator is VP64.
  • Repression of expression can be accomplished by, for example, using a nuclease free Cas9 with the appropriate tracrRNA and a targeting CRISPR array, which bind to the target nucleic acid and interfere with or repress expression of the target nucleic acid.
  • a nuclease free Cas3 polypeptide may be used with an appropriate Cascade complex and targeting CRISPR array to bind the target nucleic acid and interfere with or repress expression of the target nucleic acid.
  • a deactivated Cas3 polypeptide may also be utilized to activate transcription.
  • Cascade may also be used for repression (e.g., in the absence of Cas3).
  • a“control” as used herein may be, for example, a cell of the same species or strain(s) that has not been contacted with a composition of this invention.
  • a control may be a wild type cell or a wild type population of cells, or it may be cell or a population of cells contacted with a composition of this invention comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer) and a CRISPR array comprising a repeat-spacer-repeat sequence or a repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is not complementary to a target region in the genome of the cells of said population (i.e., non-self targeting/“scrambled spacer”).
  • PEI polyethyleneimine
  • PEO polyethylene oxide
  • a control may also be, for example, a wild type cell or a wild-type population of cells, or a population of cells contacted with a composition of this invention comprising a nucleic acid construct comprising a CRISPR array comprising a repeat-spacer-repeat sequence or a repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region that is not located adjacent to a protospacer adjacent motif (PAM) in the genome of the cell or the cells of the population.
  • PAM protospacer adjacent motif
  • A“repeat sequence” as used herein refers, for example, to any repeat sequence of a wild-type CRISPR locus or a repeat sequence of a synthetic CRISPR array that are separated by“spacer sequences” (e.g., a repeat-spacer sequence or a repeat-spacer-repeat sequence of the invention).
  • a repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR locus. Accordingly, in some embodiments, a repeat- spacer sequence or a repeat-spacer-repeat comprises a repeat that is substantially identical (e.g.
  • At least about 70% identical e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
  • a repeat from a wild-type Type II CRISPR array e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
  • a repeat sequence is 100% identical to a repeat from a wild type Type I CRISPR array, a wild type Type II CRISPR array, wild type Type III CRISPR array, wild type Type IV CRISPR array, wild type Type V CRISPR array or wild type Type VI CRISPR array.
  • a repeat sequence useful with this invention can comprise a nucleotide sequence comprising a partial repeat that is a fragment or portion of consecutive nucleotides of a repeat sequence of a CRISPR locus or synthetic CRISPR array of any of a Type I crRNA, Type II crRNA, Type III crRNA, Type IV crRNA, Type V crRNA or Type VI crRNA.
  • a CRISPR array (crRNA, crDNA) useful with this invention may be an array from any Type I CRISPR-Cas system, Type II CRISPR-Cas system, Type III CRISPR-Cas system, Type IV CRISPR-Cas system, Type V CRISPR-Cas system or a Type VI CRISPR-Cas system.
  • CRISPR array of a Type I, Type II, Type III, Type IV, Type V or Type VI CRISPR-Cas system refers to a nucleic acid construct that comprises from 5’ to 3’ a repeat-spacer-repeat sequence or comprises from 5’ to 3’ at least one repeat-spacer sequence (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 repeat-spacer sequences, and any range or value therein).
  • repeat-spacer sequence e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 repeat-spacer sequences, and any range or value therein.
  • the spacer of the prior (5’ to 3’) repeat-spacer sequence may be linked to the repeat of the following repeat-spacer (e.g., the spacer of a first repeat-spacer sequence is linked to the repeat of a second repeat-spacer sequence).
  • a CRISPR array may comprise two repeats (or two partial repeats) separated by a spacer (e.g., a repeat-spacer-repeat sequence).
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
  • “percent sequence identity” or“percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, /. e.
  • Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • percent identity may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci.
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001.
  • the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
  • Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions.
  • two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.
  • Stringent hybridization conditions and“stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra , for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes.
  • An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCE, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCE, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCE, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCE, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCE, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.
  • SDS sodium dodecyl sulfate
  • Any nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences.
  • nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in the particular species of interest.
  • A“spacer sequence” as used herein is a nucleotide sequence that is complementary to a target region in the genome (e.g., a target nucleic acid, a target DNA, a target region).
  • The“target region,” or“target nucleic acid” may also be referred to as“protospacer sequence.”
  • a protospacer sequence may be adjacent to a protospacer adjacent motif (PAM) sequence.
  • the target region may be chromosomal or extrachromosomal (e.g., a plasmid)).
  • a spacer sequence may be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target region (e.g., target nucleic acid, target DNA, target RNA).
  • the spacer sequence has 100% complementarity to the target region (e.g., target nucleic acid; target DNA/RNA).
  • the complementarity of the 3’ region of the spacer sequence to the target region e.g., target DNA/RNA
  • the overall complementarity of the spacer sequence to the target region e.g., target nucleic acid; target DNA/RNA
  • the first 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and the like, nucleotides in the 3’ region of a 20 nucleotide spacer sequence can be 100% complementary to the target region (e.g., target DNA/RNA), while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target region (e.g., target nucleic acid; target DNA/RNA).
  • the first 7 to 12 nucleotides of the spacer sequence may be 100% complementary to the target region (e.g., target nucleic acid), while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target region (e.g., target nucleic acid).
  • the first 7 to 10 nucleotides of the spacer sequence may be 100% complementary to the target region (e.g., target nucleic acid), while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target region (e.g., target nucleic acid).
  • the first 7 nucleotides (within the seed) of the spacer sequence may be 100% complementary to the target region (e.g., target nucleic acid), while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target region (e.g., target nucleic acid).
  • a“protospacer,”“target nucleic acid,”“target DNA,”“target RNA,” “target region” or“target region in the genome” refers to a region of an organism’s genome (chromosomal or plasmid) that is fully complementary or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a spacer sequence in a repeat-spacer sequence or repeat- spacer-repeat sequence.
  • a plasmid may be targeted (e.g., the target sequence is located on a plasmid).
  • a target sequence may be located in a gene, which can be in the upper (sense, coding) strand or in the bottom (antisense, non coding) strand.
  • a target sequence may be located in an intragenic region of a gene, optionally located in the upper (sense, coding) strand or in the bottom (antisense, non-coding) strand.
  • a gene that is targeted by constructs of this invention may encode a transcription factor or a promoter.
  • a gene that is targeted may encode non-coding RNA, including, but not limited to, eukaryotic mi RNA, siRNA, piRNA (piwi-interacting RNA) and IncRNA (long non-coding RNA).
  • a target sequence may be located in an intergenic region, optionally in the upper (plus) strand or in the bottom (minus) strand.
  • a target sequence may be located in a intergenic region wherein the DNA is cleaved and a gene inserted that may be expressed under the control of the promoter of the previous open reading frame.
  • a target sequence may be located on a mobile genetic element (e.g., a transposon, a plasmid, a bacteriophage element (e.g., Mu)).
  • a mobile genetic element e.g., a transposon, a plasmid, a bacteriophage element (e.g., Mu)
  • mobile genetic elements located in the chromosome or transposons may be targeted to force the mobile elements to jump out of the chromosome.
  • a target region may be about 10 to about 40 consecutive nucleotides in length located immediately adjacent to a PAM sequence (PAM sequence located immediately 3’ of the target region) in the genome of the organism (e.g., Type I CRISPR-Cas systems and Type II CRISPR-Cas systems).
  • PAM sequence located immediately 3’ of the target region
  • the PAM is on the alternate side of the protospacer (the 5’ end).
  • PAM is on the alternate side of the protospacer (the 5’ end).
  • Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). Guide structures and PAMs are described in by R. Barrangou ( Genome Biol. 16:247 (2015)).
  • a target nucleic acid e.g., target DNA/RNA
  • a protospacer adjacent motif PAM
  • the CRISPR array is a Type I CRISPR array and the target nucleic acid (protospacer) is located adjacent to the 3’ end of a PAM (PAM is 5’ to the protospacer).
  • the CRISPR array is a Type II CRISPR array and the target nucleic acid (e.g., target DNA/RNA) is located adjacent to the 5’ end of the PAM (PAM is 3’ to the protospacer).
  • the CRISPR array is a Type V CRISPR array, and the target nucleic acid (e.g., target DNA/RNA) is typically located adjacent to the 5’ end of the PAM (PAM is 3’ to the protospacer) similar to Type II.
  • a target nucleic acid useful with a Type V CRISPR-Cas system may be located adjacent to the 3’ end of the PAM (PAM is 5’ to the protospacer), similar to Type I.
  • the CRISPR array is a Type III CRISPR array or a Type VI CRISPR array and the target nucleic acid (e.g., target DNA RNA) is not adjacent to a PAM but instead may be located within the target RNA.
  • “adjacent” can mean immediately adjacent to (5’ or 3’ depending on the system) or it can mean one to seven nucleotides between the PAM and the protospacer.
  • a PAM is typically immediately upstream of the protospacer.
  • a PAM is typically one to two nucleotides downstream of the protospacer but can be from about one to seven nucleotides downstream of the protospacer (e.g., 1, 2, 3, 4, 5, 6, 7 nucleotides; e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 3 to 5, 3 to 7, 4 to 7, 5 to 7 nucleotides downstream of the protospacer).
  • a PAM for a Type II-C CRISPR system may be 5 to 7 nucleotides downstream of the protospacer.
  • a PAM is typically one to two nucleotides downstream of the protospacer.
  • a protospacer adjacent motif may comprise, consist essentially of, or consist of a nucleotide sequence of 5’-NAA-3’ and/or 5’ -AAA-3’, 5’-NOG S’, 5’-NGAAA-3’, 5’-NNG-3’, 5’-NGA-3’, 5’-NTAA-3’, 5’-NTG-3’, 5’-NNC-3’, 5’- NNAAC-3’, 5’-AGA-3’, 5’ -NNN ANNA-3’ , 5’-NNANAA-3’, 5’-NNAAAA-3’, and/or 5’- AAAA-3’ .
  • a target region can be randomly selected or can be specifically selected.
  • a target nucleic aci d/region may be within an essential gene or it may be within a non-essential gene.
  • a randomly selected target region may be selected from any at least 10 consecutive nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
  • the target region may be about 10 to about 20 consecutive nucleotides, about 10 to about 30 consecutive nucleotides, and/or about 10 to about 40 consecutive nucleotides and the like, or any range or value therein, located immediately adjacent to a protospacer adjacent motif (PAM) sequence in a genome.
  • PAM protospacer adjacent motif
  • specifically selecting a target region comprises specifically selecting a target region from a gene, open reading frame, a putative open reading frame or an intergenic region comprising at least about 10 to about 40 consecutive nucleotides immediately adjacent to a PAM sequence in a genome.
  • a "trans-activating CRISPR (tracr) nucleic acid” or “tracr nucleic acid” as used herein refers to any tracr RNA (or its encoding DNA).
  • a tracr nucleic acid comprises from 5’ to 3’ a lower stem, an upper stem, a bulge, a nexus hairpin and terminal hairpins (See, Briner et al. (2014) Molecular Cell. 56(2):333-339).
  • a trans-activating CRISPR (tracr) nucleic acid functions in hybridizing to the repeat portion of mature or immature crRNAs, recruits Cas9 protein to the target site, and may facilitate the catalytic activity of Cas9 by inducting structural rearrangement.
  • the functional composition of tracrRNA molecules is listed above. Sequences for tracrRNAs are specific to the CRISPR-Cas system and can be variable. Any tracr nucleic acid, known or later identified, can be used with this invention.
  • the phrase“substantially identical,” or“substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that have about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
  • nucleic acid molecule of the invention may be about 80% to about 100% identical over at least about 10 nucleotides consecutive nucleotides to about 25 consecutive nucleotides of a reference nucleic acid sequence (e.g., a repeat sequence).
  • sequences of the invention can be about 80% to about 100% identical to about 15 consecutive nucleotides to about 25 consecutive nucleotides of a reference sequence. In some embodiments, sequences of the invention can be at least 70% identical over at least 18 consecutive nucleotides. In other embodiments, the sequences can be at least 85% identical over at least 20 consecutive nucleotides of a reference sequence. In still other embodiments, the nucleic acid molecule of the invention can be 100% identical over at least 15 consecutive nucleotides of a reference sequence. In a further embodiment, the sequences are substantially identical (e.g., about 70% to about 99%) over the entire length of a coding region of a reference sequence.
  • a substantially identical nucleotide or polypeptide sequence performs substantially the same function as the nucleotide or polypeptide sequence to which it is substantially identical to (e.g., the function or activity of a crRNA, tracr nucleic acid, repeat sequence, Cas nuclease (nickase, DNA, RNA and/or PAM recognition and binding), Cas9, Cas3, Cas3’, Cas3”, Casl3 (formerly C2c2), Cascade complex, and the like, or any other Type I, Type II, Type III, Type IV, Type V or Type VI CRISPR-Cas polynucleotide or polypeptide).
  • a crRNA tracr nucleic acid, repeat sequence, Cas nuclease (nickase, DNA, RNA and/or PAM recognition and binding), Cas9, Cas3, Cas3’, Cas3”, Casl3 (formerly C2c2), Cascade complex, and the like, or any other Type I, Type II
  • the phrase “substantially complementary,” or “substantial complementarity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue complementary, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantial complementarity can refer to two or more sequences or subsequences that have at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 96, 97, 98, or 99% complementarity (e.g., about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99% or more, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99% or more, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99% or more, about 95% to about 97%, about 95% to about 98%, about 95% to about 99% or more).
  • complementarity e.g., about 80% to about 90%, about 80% to about 95%,
  • nucleic acid molecules for transforming a cell may comprise/encode CRISPR-Cas system nucleic acids and/or polypeptides, thereby editing or modifying the genome of the cell or killing the cell.
  • the composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleic acid molecules, which may be complexed with a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a single polyalkyleneimine-alkoxylene polymer e.g., PEI-PEO polymer
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleic acid molecules e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleic acid molecules.
  • a single nucleic acid molecule is complexed with two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers).
  • two or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
  • polyalkyleneimine-alkoxylene polymers e.g., PEI-PEO polymers
  • a method of editing/modifying a target nucleic acid in the genome of a cell (e.g., a target cell, host cell; e.g., cell of an organism) comprising introducing into the cell a first composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer) and a CRISPR-Cas array (CRISPR array, crRNA, crDNA), wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to a target nucleic acid in the genome of the cell, and at least one additional component of a CRISPR-Cas system, optionally wherein the at least one additional component is complexed with the first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkylenei
  • a first polyalkyleneimine-alkoxylene polymer e.
  • editing/modifying a target nucleic acid comprises altering the genotype of the cell or one or more cells in a population.
  • the present invention further provides a method for site-specific cleavage of a target nucleic acid (e.g., target DNA/RNA) in the genome of a cell (e.g., a target cell; cell of a target organism), comprising introducing into the cell a first composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer) and a CRISPR-Cas array (CRISPR array, crRNA, crDNA), wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to a target nucleic acid in the genome of the cell, and at least one additional component of a CRISPR-Cas system, optionally wherein the at least one additional component is complexed with the first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxy
  • the at least one additional component of a CRISPR-Cas system may be introduced in a second composition, optionally wherein the at least one additional component is complexed with the first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxylene polymer (e.g., a second PEI- PEO polymer).
  • first polyalkyleneimine-alkoxylene polymer e.g., a first PEI-PEO polymer
  • a second polyalkyleneimine-alkoxylene polymer e.g., a second PEI- PEO polymer
  • a CRISPR array useful with a method for site-specific cleavage of a target nucleic acid (e.g., target DNA, target RNA) in the genome of a cell and/or a method of editing/modifying a target nucleic acid in the genome of a cell may be, for example, a Type I CRISPR array, Type II CRISPR array, Type III CRISPR array, Type IV CRISPR array, Type V CRISPR array or Type VI CRISPR array.
  • the CRISPR array may comprise at least one repeat sequence linked to the 5’ end and/or the 3’ end of the at least one spacer sequence.
  • the CRISPR array may comprises a repeat sequence linked to the 5’ end and to the 3’ end of the at least one spacer sequence.
  • a composition useful in a method for site-specific cleavage of a target nucleic acid (e.g., target DNA/RNA) in the genome of a cell (e.g., a target cell, host cell) and/or a method of editing/modifying a target nucleic acid in the genome of a cell may further comprise at least one additional component of a CRISPR-Cas system.
  • the at least one additional component of a CRISPR-Cas system may be complexed with a first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxylene polymer (e.g., a second PEI-PEO polymer).
  • a first polyalkyleneimine-alkoxylene polymer e.g., a first PEI-PEO polymer
  • a second polyalkyleneimine-alkoxylene polymer e.g., a second PEI-PEO polymer
  • the method may further comprise introducing into the cell a second composition (e.g., a separate composition from that comprising the polymer and CRISPR array) comprising a polyalkyleneimine-alkoxylene polymer and at least one additional component of a CRISPR-Cas system, optionally wherein the at least one additional component may be complexed with the polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer).
  • a second composition e.g., a separate composition from that comprising the polymer and CRISPR array
  • a second composition comprising a polyalkyleneimine-alkoxylene polymer and at least one additional component of a CRISPR-Cas system, optionally wherein the at least one additional component may be complexed with the polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer).
  • the polyalkyleneimine-alkoxylene polymer in the second composition may be the same polyalkyleneimine-alkoxylene polymer that is in the first composition (e.g., separate composition comprising the polymer and CRISPR array) or it may be a different polyalkyleneimine-alkoxylene polymer.
  • An at least one additional component of a CRISPR-Cas system useful with the methods of the invention includes but is not limited to a nucleic acid encoding a CRISPR-Cas nuclease, a trans-encoded CRISPR (tracr) nucleic acid, and/or a nucleic acid encoding an additional CRISPR-Cas polypeptide.
  • the at least one additional component is a tracr nucleic acid
  • the tracr nucleic acid and CRISPR array may be introduced as a chimeric nucleic acid molecule (e.g., single guide, sgDNA).
  • a CRISPR-Cas nuclease useful with a method of the invention may be, for example, a Type I CRISPR-Cas nuclease, a Type II CRISPR-Cas nuclease, a Type III CRISPR-Cas nuclease, a Type IV CRISPR-Cas nuclease, Type V CRISPR-Cas nuclease and/or a Type VI CRISPR-Cas nuclease.
  • the Type II CRISPR-Cas nuclease is a Cas9 nuclease
  • the Type I nuclease is a Cas3 nuclease
  • the Type III nuclease is a CaslO nuclease
  • the Type IV nuclease is a Csf4 nuclease
  • Type V CRISPR-Cas nuclease is a Casl2 nuclease and/or the Type V nuclease is a Casl3 nuclease.
  • Non-limiting examples of an additional CRISPR-Cas polypeptide may be a Type I CRISPR associated complex for antiviral defense complex (Cascade complex) polypeptide, a Type III Csm complex polypeptide, a Type III Csr complex polypeptide, a Type IV polypeptide, Type V polypeptide and/or a Type VI polypeptide.
  • Cascade complex a Type I CRISPR associated complex for antiviral defense complex
  • Type III Csm complex polypeptide a Type III Csr complex polypeptide
  • Type IV polypeptide Type V polypeptide and/or a Type VI polypeptide.
  • a method for transcriptional control of a target nucleic acid in the genome of a cell (e.g., a target cell, a host cell)
  • the method comprising introducing into the cell a composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer), a Type II CRISPR-Cas array (CRISPR array), and a nucleic acid encoding a deactivated Cas9 nuclease, wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to the target nucleic acid in the genome of the cell, thereby controlling the transcription of the target nucleic acid in the genome of the cell.
  • a first polyalkyleneimine-alkoxylene polymer e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer
  • PEO polyethyleneimine
  • CRISPR array Type
  • a deactivated Cas9 nuclease can comprise a mutated HNH motif that lacks HNH nickase activity and/or a mutated RuvC motif that lacks RuvC nickase activity.
  • the CRISPR array and nucleic acid encoding a deactivated Cas9 nuclease may be complexed with the same or a different polyalkyleneimine-alkoxylene polymer and may be provided in the same or different compositions (e.g., a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth composition and the like).
  • the Type II CRISPR array may comprise at least one repeat sequence linked to the 5’ end and/or the 3’ end of the at least one spacer sequence. In some embodiments, the Type II CRISPR array may comprises a repeat sequence linked to the 5’ end and to the 3’ end of the at least one spacer sequence.
  • an exogenous tracr nucleic acid may be introduced into a cell of a eukaryotic organism either in the same composition or in a separate composition from that which is used to introduce the CRISPR array.
  • a composition introduced into a cell may further comprise a tracr nucleic acid, optionally wherein the tracr nucleic acid is complexed with a first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxylene polymer (e.g., a second PEI-PEO polymer) (e.g., complexed with the same or a different polymer).
  • a first polyalkyleneimine-alkoxylene polymer e.g., a first PEI-PEO polymer
  • a second polyalkyleneimine-alkoxylene polymer e.g., a second PEI-PEO polymer
  • a second composition comprising a tracr nucleic acid and a polyalkyleneimine-alkoxylene polymer may be introduced into a cell, optionally wherein the tracr nucleic acid may be complexed with the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • the polyalkyleneimine-alkoxylene polymer in the second composition may be the same polyalkyleneimine-alkoxylene polymer that is in the first composition (e.g., the composition comprising a polymer and a CRISPR array) or it may be a different polyalkyleneimine- alkoxylene polymer.
  • the CRISPR array may form a CRISPR array- tracr complex with the tracr nucleic acid.
  • the CRISPR array-tracr complex can then bind to the target nucleic acid and recruit a deactivated Cas9 nuclease expressed from a nucleic acid encoding a deactivated Cas9 nuclease, thereby controlling the transcription of the target nucleic acid in the genome of the cell.
  • a CRISPR array and tracr nucleic acid may be introduced into a cell or a population of cells as a single guide nucleic acid (e.g., sgDNA, sgRNA).
  • a single guide nucleic acid comprises a tracr nucleic acid covalently linked to a CRISPR RNA by intervening nucleotides ("linker nucleotides” or "a linker nucleotide sequence"), wherein the 5’ end of the tracr nucleic acid is covalently linked to the 3’ end of the CRISPR RNA via the linker nucleotide sequence.
  • a linker nucleotide sequence may comprise 3 or more nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 134, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more nucleotides, and any range or value therein).
  • nucleotides e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 134, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47
  • the linker nucleotide sequence may be about 3 to about 100 nucleotides, about 4 to about 50 nucleotides, about 4 to about 30 nucleotides, about 5 to about 50 nucleotides, about 5 to about 30 nucleotides and the like.
  • a composition comprising a polyalkyleneimine-alkoxylene polymer and a single guide may be introduced into a cell (or a population of cells), wherein the single guide may be complexed with the polyalkyleneimine-alkoxylene polymer.
  • a method for transcriptional control of a target nucleic acid in the genome of a cell (e.g., a target cell, a host cell)
  • the method comprising introducing into the cell a composition comprising a first polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer), a Type I CRISPR-Cas array (CRISPR array, crRNA, crDNA), and a nucleic acid encoding a deactivated Cas3, wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to the target nucleic acid in the genome of the cell, thereby controlling the transcription of the target nucleic acid.
  • a first polyalkyleneimine-alkoxylene polymer e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer
  • PEO polyethyleneimine
  • CRISPR array Type I CRISPR-C
  • a deactivated Cas3 can comprise a mutated histidine-aspartate (HD) nuclease activity and/or a mutated Superfamily 2 (SF2) helicase activity.
  • Cascade may be introduced in the same or a different composition of the invention.
  • a first polyalkyleneimine-alkoxylene polymer e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer
  • CRISPR array Type I CRISPR-Cas array
  • Cascade may be introduced in the same or a separate composition of the invention from the composition introducing the CRISPR array.
  • a deactivated Cas3 can comprise a mutated histidine-aspartate (HD) nuclease activity and/or a mutated Superfamily 2 (SF2) helicase activity.
  • HD histidine-aspartate
  • SF2 mutated Superfamily 2
  • the Type I CRISPR array may comprise at least one repeat sequence linked to the 5’ end and/or the 3’ end of the at least one spacer sequence. In some embodiments, the Type I CRISPR array may comprises a repeat sequence linked to the 5’ end and to the 3’ end of the at least one spacer sequence.
  • a composition comprising a Type I CRISPR array may further comprise a nucleic acid encoding a Type I Cascade complex, optionally wherein the nucleic acid encoding a Type I Cascade complex may be complexed with a first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxylene polymer (e.g., a second PEI-PEO polymer) (e.g., the composition comprising the nucleic acid encoding a Type I Cascade complex may comprise the same or a different polymer from that which is in the composition comprising the CRISPR array).
  • a first polyalkyleneimine-alkoxylene polymer e.g., a first PEI-PEO polymer
  • a second polyalkyleneimine-alkoxylene polymer e.g., a second PEI-PEO polymer
  • a second composition comprising a polyalkyleneimine-alkoxylene polymer and a nucleic acid encoding a Type I Cascade complex may be introduced into the cell, optionally wherein the nucleic acid encoding a Type I Cascade complex may be complexed with the polyalkyleneimine-alkoxylene polymer.
  • the polyalkyleneimine-alkoxylene polymer in a second composition may be the same polyalkyleneimine- alkoxylene polymer that is in the first composition (or second composition, third composition, fourth composition and the like) or it may be a different polyalkyleneimine- alkoxylene polymer (e.g., a second PEI-PEO polymer).
  • the polynucleotides encoding the polypeptides comprising a Type I Cascade complex may be introduced together in the same composition (on the same or different nucleic acid molecules) or separately in one or more different compositions with the same or a different polyalkyleneimine-alkoxylene polymer.
  • the Type I Cascade complex may form a CRISPR array- Type I Cascade complex with the CRISPR array.
  • the CRISPR array-tracr complex can then bind to the target nucleic acid (e.g., target DNA/RNA) and recruit a deactivated Cas3 nuclease expressed from a nucleic acid encoding a deactivated Cas3 nuclease, thereby controlling the transcription of the target nucleic acid in the genome of the host cell.
  • the target nucleic acid e.g., target DNA/RNA
  • the present invention further provides methods for killing cells, including one or more cells in a population.
  • a population comprises different types of cells one or more cells of a particular type (having a unique target nucleic acid) may be killed using the compositions and methods of this invention. This may be accomplished by designing the spacer of the CRISPR array to target one or more target nucleic acids unique to the cells to be killed in the population, thereby killing only those cells comprising that target nucleic acid.
  • a method of killing a cell comprising introducing into the cell a first composition comprising a first polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer and a CRISPR-Cas array (CRISPR array, crRNA, crDNA), wherein the CRISPR array comprises at least one spacer sequence having substantial complementarity to a target nucleic acid (e.g., target region; target DNA/RNA) in the genome of the cell, and at least one additional component of a CRISPR-Cas system, optionally wherein the at least one additional component is complexed with the first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxylene polymer (e.g., a second PEI-PEO polymer), thereby killing the cell.
  • a first polyalkyleneimine-alkoxylene polymer e.g., a first PEI-PEO polymer
  • a CRISPR array useful in a method for killing cells may be, for example, a Type I CRISPR array, Type II CRISPR array, Type III CRISPR array, Type IV CRISPR array, Type V CRISPR array or Type VI CRISPR array.
  • the CRISPR array may comprise at least one repeat sequence linked to the 5’ end and/or the 3’ end of the at least one spacer sequence.
  • the Type II CRISPR array may comprises a repeat sequence linked to the 5’ end and to the 3’ end of the at least one spacer sequence.
  • the at least one additional component of a CRISPR-Cas system may be complexed with a first polyalkyleneimine-alkoxylene polymer (e.g., a first PEI-PEO polymer) or a second polyalkyleneimine-alkoxylene polymer (e.g., a second PEI-PEO polymer).
  • a first polyalkyleneimine-alkoxylene polymer e.g., a first PEI-PEO polymer
  • a second polyalkyleneimine-alkoxylene polymer e.g., a second PEI-PEO polymer
  • the at least one additional component of a CRISPR-Cas system may be introduced in a separate composition from that comprising a CRISPR array (e.g., a second composition), wherein the second or separate composition comprises a polyalkyleneimine-alkoxylene polymer and at least one additional component of a CRISPR- Cas system, optionally wherein the at least one additional component may be complexed with the polyalkyleneimine-alkoxylene polymer.
  • the polyalkyleneimine- alkoxylene polymer in a second or separate composition may be the same polyalkyleneimine- alkoxylene polymer that is present in the first composition (e.g., the composition comprising the CRISPR array) or it may be a different polyalkyleneimine-alkoxylene polymer (e.g., a second PEI-PEO polymer).
  • An at least one additional component of a CRISPR-Cas system useful with the methods of the invention for killing a cell includes, but is not limited to, a nucleic acid encoding a CRISPR-Cas nuclease, a trans-encoded CRISPR (tracr) nucleic acid, and/or a nucleic acid encoding an additional CRISPR-Cas polypeptide.
  • the at least one additional component is a tracr nucleic acid
  • the tracr nucleic acid and CRISPR array may be introduced as a single guide.
  • a CRISPR-Cas nuclease useful with the methods of the invention may be, for example, a Type I CRISPR-Cas nuclease, a Type II CRISPR-Cas nuclease, a Type III CRISPR-Cas nuclease, a Type IV CRISPR-Cas nuclease, a Type V CRISPR-Cas nuclease and/or a Type VI CRISPR-Cas nuclease.
  • the Type II CRISPR-Cas nuclease is a Cas9 nuclease
  • the Type I nuclease is a Cas3 nuclease
  • the Type III nuclease is a CaslO nuclease
  • the Type IV nuclease is a Csf4 nuclease
  • the Type V nuclease is a Casl2 nuclease
  • the Type VI nuclease is a Casl3 nuclease.
  • Non-limiting examples of an additional CRISPR-Cas polypeptide may be a Type I CRISPR associated complex for antiviral defense complex (Cascade complex) polypeptide, a Type III Csm complex polypeptide, a Type III Csr complex polypeptide, a Type IV polypeptide, a Type V polypeptide and/or a Type VI polypeptide.
  • Cascade complex CRISPR associated complex for antiviral defense complex
  • a Cas9 polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to an amino acid sequence of a Cas9 nuclease.
  • Exemplary Cas9 nucleases useful with this invention can be any Cas9 nuclease known to catalyze DNA cleavage in a CRISPR-Cas system.
  • Cas9 nucleases comprise a HNH motif and a RuvC motif (See, e.g., WO2013/176772; WO/2013/188638).
  • a functional fragment of a Cas9 nuclease can be used with this invention.
  • CRISPR-Cas systems and groupings of Cas9 nucleases are well known in the art and include, for example, a Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, a Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, and a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases.
  • Additional Cas9 nucleases include, but are not limited to, those of Lactobacillus curvatus CRL 705.
  • Cas9 nucleases useful with this invention include, but are not limited to, a Cas9 from Lactobacillus animalis KCTC 3501, Lactobacillus farciminis WP 010018949.1 and other previously characterized Lactobacillus Cas9 nucleases such as L. casei, L. jensenii, L. gasseri and L. pentosus (see Crawley et ah, 2018).
  • a Type I polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
  • a Type I Cascade polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%,
  • Cas7 Csa2
  • Cas8al Csxl3
  • Cas8a2 Csx9
  • Cas5 Csa5
  • Cas6a Cas6b
  • Cas8b Cas7
  • Cas7 Cash2
  • Cas5d Cas8c
  • Cas7 Casd2
  • CaslOd Casc3
  • Csc2 Cscl
  • Cas6d Csel (CasA), Cse2 (CasB
  • Cas7 CasC
  • Cas5 Case2
  • CasE Cas6e
  • Cys2 Cas7 (Cys3)
  • Cas6f Cas6 and/or Cas4
  • Type I CRISPR-Cas systems are well known in the art and include, for example, Archaeoglobus fulgidus comprises an exemplary Type I-A CRISPR-Cas system, Clostridium kluyveri DSM 555 comprises an exemplary Type I-B CRISPR-Cas system, Bacillus halodurans C-125 comprises an exemplary Type I-C CRISPR-Cas system, Cyanothece sp.
  • PCC 802 comprises an exemplary Type I-D CRISPR-Cas system
  • Escherichia coli K-12 and Lactobacillus crispatus comprise exemplary Type I-E CRISPR-Cas systems
  • Geobacter sulfurreducens comprises an exemplary Type I-U CRISPR-Cas system
  • Yersinia pseudotuberculosis YPIII comprises an exemplary Type I-F CRISPR-Cas system.
  • a Type II polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to an amino acid sequence of a Cas9.
  • Type II CRISPR- Cas systems well known in the art and include, for example, Legionella pneumophila str.
  • Streptococcus thermophilus CNRZ1066 both SthelCas9 and Sthe3Cas9
  • Neisseria lactamica 020-06 as well as Streptococcus pyogenes (SpyCas9j, Neisseria meningitidis (NmeCas9), Staphylococcus aureus (SauCas9), and Lactobacillus gasseri (LgaCas9).
  • a Type III polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to an amino acid sequence of a Cas6, Cas 10 (or Csml), Csm2, Csm3, Csm4, Csm5, and Csm6, Cmrl, CaslO (or Cmr2), Cmr3, Cmr4, Cmr5, and Cmr6, Cas7, CaslO, Cas7 (Csm3), Cas5 (Csm4), Cas7 (Csm5), Csm6, Cas7 (Cmrl), Cas5 (Cmr3), Cas
  • Type III CRISPR-Cas systems are well known in the art and include, for example, Staphylococcus epidermidis RP62A, which comprises an exemplary Type III-A CRISPR-Cas system, Pyrococcus furiosus DSM 3638, which comprises an exemplary Type III-B CRISPR-Cas system, Methanothermobacter thermautotrophicus str. Delta H, which comprises an exemplary Type III-C CRISPR-Cas system, and Roseiflexis sp. Rs-1, which comprises an exemplary Type III-D CRISPR-Cas system.
  • a Type IV polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to an amino acid sequence of a Csf4 (dinG), Csfl, Cas7 (Csf2) and/or Cas5 (csfl).
  • Type IV CRISPR-Cas systems are well known in the art, for example, Acidithiobacillus ferrooxidans ATCC 23270 comprises an exemplary Type IV CRISPR-Cas system.
  • a Type V polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to an amino acid sequence of a Cpfl (Casl2a), Casl, Cas2, or Cas4.
  • Type V CRISPR-Cas systems are well known in the art and include, for example, Francisella cf. novicida Fxl comprises an exemplary Type V CRISPR-Cas system.
  • a Type VI polypeptide useful with this invention comprises at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to an amino acid sequence of a Casl3.
  • 70% identity e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like
  • Type V CRISPR- Cas systems are well known in the art and include, for example, Leptotrichia shahii, Leptotricia buccalis and Listeria seeligeri comprise an exemplary Type VI CRISPR-Cas system.
  • more than one composition may be introduced into a cell (e.g., a target cell, a host cell).
  • a cell e.g., a target cell, a host cell.
  • the compositions may be introduced into the cell simultaneously, separately, and/or sequentially, in any combination.
  • at least two of the more than one compositions may be introduced into the cell as a mixture comprising the first and second compositions.
  • first composition and second composition when introduced into the cell, they may be introduced as a mixture comprising the first and second compositions, or when a first composition, a second composition and a third composition are introduced into the cell, the first, second and third compositions may be introduced as a mixture comprising the first, second, and third compositions.
  • the weight ratio of the CRISPR array, nucleic acid encoding a CRISPR-Cas nuclease, tracr nucleic acid, single guide nucleic acid, and/or the nucleic acid encoding an additional CRISPR-Cas polypeptide to the polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) in a composition of the invention may be about 1 : 10 to about 1 : 10,000 (nucleic acid: polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer)) in the composition.
  • the amount of the CRISPR array, nucleic acid encoding a CRISPR nuclease, tracr nucleic acid, single guide, and/or nucleic acid encoding an additional CRISPR polypeptide present in a composition may be about 0.1, 0.5, 1, 5, 10, 15, 20, or 25 nanograms per microgram of the polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) to about 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nanograms per microgram of the polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer).
  • the polyalkyleneimine-alkoxylene polymer e.g., PEI-PEO polymer
  • the polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) in a composition of the invention is cationic.
  • the composition of the invention comprises a CRISPR array, nucleic acid encoding a CRISPR nuclease, tracr nucleic acid, single guide, and/or nucleic acid encoding an additional CRISPR polypeptide electrostatically and/or covalently bound to a portion of the polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer).
  • the polyalkyleneimine-alkoxylene polymer comprises 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of the polyalkyleneimine (e.g., PEI or polypropyleneimine) in the polyalkyleneimine-alkoxylene polymer, optionally wherein the polyalkyleneimine- alkoxylene polymer may comprise 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of the polyalkyleneimine (e.g., PEI or polypropyleneimine) in the polyalkyleneimine-alkoxylene polymer.
  • alkoxylene units e.g., ethylene oxide units
  • At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the primary amines of the polyalkyleneimine (e.g., PEI or polypropyleneimine) of the polyalkyleneimine-alkoxylene polymer in a composition of the invention are functionalized with at least one alkoxylene unit (e.g., ethylene oxide unit), optionally wherein one or more primary amines of the polyalkyleneimine are functionalized with at least one alkoxylene unit to form the polyalkyleneimine-alkoxylene.
  • alkoxylene unit e.g., ethylene oxide unit
  • a polyalkyleneimine-alkoxylene polymer of the composition comprises PEI in an amount of about 1 to about 40 % by weight of the polymer and comprises PEO in an amount of about 60 to about 99% by weight of the polymer.
  • Polyethyleneimines such as Lupasol ® P e.g., 50% concentrated in water, 750,000 MW
  • Lupasol ® G100 e.g., 50% in water, 5,000 MW
  • Lupasol ® HF e.g., 56% in water, 25 000 MW
  • PEIs were in water and used as obtained.
  • Lupasol ® P and Lupasol ® G100 were used in 50% water and Lupasol ® HF was used in 56% water.
  • Dimethyl sulfate, n-bromobutane, 1-bromooctane, 1- bromododecane, and 1-bromooctadecane were each obtained from Sigma Aldrich. The purity of each halogenated hydrocarbon was, at minimum, greater than 95%.
  • the molecular weight of the polyethyleneimines were determined by GPC against PEG standards and reported in g/mol.
  • the molecular weight (as reported in g/mol) of the PEI-alkoxylates were determined by hydroxyl value and extrapolated by the average molar equivalent of primary amine per polyethylamine.
  • the acid value was measured by titrating 1 gram of PEI-alkoxylate with a 0.1 N KOH solution in methanol (as reported mg KOH/lg of material)
  • the amine functionality was measured by carbon- 13 NMR; taking the average area of each amine region (i.e., 35 - 45 PPM for primary amines) over the total amine region (35 - 60 PPM). The results for each amine type were then reported as the ratio of primary, secondary and tertiary amines.
  • the charge density of the PEIs (unless otherwise stated) were measured from the dried substance at pH 4.5
  • the hydroxyl number was measured by reacting 1 g of PEI-alkoxylate with acetic anhydride in the presence of DMAP (Dimethyl aminopyridine) and back titrating with 0.5 N KOH solution in Methanol.
  • DMAP Dimethyl aminopyridine
  • PEI polyethyleneimine
  • Agitation was set to 350 rpm.
  • the reactor was sealed and nitrogen purged (3 times at 6.2 bar) before heating. Pressure was adjusted to 1.4 bar with nitrogen and the reactor heated to 120°C.
  • Ethylene oxide (6.84 molar equivalents) was added over 2 hours and reacted to constant pressure.
  • the reactor was cooled to 90°C and an aqueous solution including KOH in an amount of 45 % by weight of the aqueous solution was added to the reactor in an amount of 0.15 wt% of the mixture in the reactor. Water was stripped for 1 hour at 120°C and 0.13 bar.
  • Ethylene oxide (33.86 molar equivalent) was fed into the reactor at a rate of 4 g/min. The rate was increased to 6 g/min as quickly as temperature control and pressure allowed. Total addition time was 5 hrs. After the ethylene oxide addition was completed, reacted to constant pressure for 2 additional hours. The reactor was vented and cooled to 85°C before discharging. By this procedure, an ethoxylated polyethyleneimine was obtained as a brown liquid or as a solid. Degree of ethoxylation was determine by OH#. Average molecular weight was determined by GPC in water/THF. Amine functionality confirmed by 13 C NMR.
  • Table 3 Summary of PEI polymers used and PEI-PEO polymers.
  • step 2 Take about 660 m ⁇ of polymer solution from step 1, transfer to 1.5 mL micro-centrifuge tube.
  • the parameters of these protocols may be modified to include variations in the ratio of nucleic acid to polymer; the type of nucleic acid used; as well as adjusted for use with cells from different species.
  • Polymers 1, 3, 7, and 9 were evaluated for their effect on plant cell viability, penetration, and DNA delivery efficiency.
  • Polymers 7 an 9 have a larger core than polymers 1 and 3 and a greater cationic charge. Polymers 1 and 7 have a higher cationic charge than polymers 3 and 9.
  • Protoplasts are naked plant cells which have had their cell wall removed by either mechanical or enzymatic means.
  • protoplasts derived from several plant species have been widely used to study cellular processes and elucidate molecular mechanisms underlying pathways involved in plant physiology, immunity, growth and development.
  • the transformation potential of the polymers for plasmid DNA delivery into wheat protoplast cells was assessed.
  • cells were transfected with a GFP-expressing plasmid and assayed for fluorescence 2 days post treatment. Initially, each of the polymers was used at the highest non-lethal concentration as determined in abovementioned experiment and mixed with 10 pg of plasmid DNA.
  • Control cells were transfected using polyethylene glycol (PEG) following our standard transfection protocol. The transfection efficiency was calculated as the number of GFP-positive cells divided by the total number of living cells at the end of the experiments.
  • PEG polyethylene glycol
  • the polymers will be particularly useful in transforming intact plant cells (e.g., cells with cell walls, e.g., plants and plant tissues). This method should provide a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium and the like.

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Abstract

La présente invention concerne de manière générale des méthodes de modification du génome d'un organisme, comprenant la mise en contact du génome de l'organisme avec une composition comprenant un acide nucléique CRISPR-Cas et un polymère de polyalkylèneimine-alcoxylène tel qu'un polymère de polyéthylèneimine (PEI)-oxyde de polyéthylène (PEO). La présente invention concerne en outre des méthodes d'utilisation des composition de l'invention pour tuer des cellules.
PCT/US2020/039063 2019-06-24 2020-06-23 Méthodes et compositions permettant de modifier des acides nucléiques et de tuer des cellules Ceased WO2020263782A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007133812A2 (fr) * 2005-12-30 2007-11-22 Philadelphia Health & Education Corporation, D/B/A Drexel University College Of Medicine Supports améliorés destinés à distribuer des agents d'acides nucléiques à des cellules et à des tissus
WO2015173824A1 (fr) * 2014-05-14 2015-11-19 Alex Levitzki Management And Holdings Ltd Vecteurs améliorés a base de polyéthylèneimine polyéthylèneglycol
US20170028083A1 (en) * 2014-04-08 2017-02-02 North Carolina State University Methods and Compositions for RNA-Directed Repression of Transcription Using CRISPR-Associated Genes
WO2017053312A1 (fr) * 2015-09-21 2017-03-30 The Regents Of The University Of California Compositions et méthodes de modification d'acides nucléiques cibles
WO2017053713A1 (fr) * 2015-09-25 2017-03-30 Tarveda Therapeutics, Inc. Compositions et méthodes pour l'édition génomique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007133812A2 (fr) * 2005-12-30 2007-11-22 Philadelphia Health & Education Corporation, D/B/A Drexel University College Of Medicine Supports améliorés destinés à distribuer des agents d'acides nucléiques à des cellules et à des tissus
US20170028083A1 (en) * 2014-04-08 2017-02-02 North Carolina State University Methods and Compositions for RNA-Directed Repression of Transcription Using CRISPR-Associated Genes
WO2015173824A1 (fr) * 2014-05-14 2015-11-19 Alex Levitzki Management And Holdings Ltd Vecteurs améliorés a base de polyéthylèneimine polyéthylèneglycol
WO2017053312A1 (fr) * 2015-09-21 2017-03-30 The Regents Of The University Of California Compositions et méthodes de modification d'acides nucléiques cibles
WO2017053713A1 (fr) * 2015-09-25 2017-03-30 Tarveda Therapeutics, Inc. Compositions et méthodes pour l'édition génomique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KOONIN ET AL.: "Origins and evolution of CRISPR-Cas systems", PHILOS TRANS R SOC LOND B BIOL SCI, vol. 374, no. 1772, 13 May 2019 (2019-05-13), pages 1 - 16, XP055674517, DOI: 10.1098/rstb.2018.0087 *

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