WO2023030534A1 - 改进的引导编辑系统 - Google Patents

改进的引导编辑系统 Download PDF

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WO2023030534A1
WO2023030534A1 PCT/CN2022/117258 CN2022117258W WO2023030534A1 WO 2023030534 A1 WO2023030534 A1 WO 2023030534A1 CN 2022117258 W CN2022117258 W CN 2022117258W WO 2023030534 A1 WO2023030534 A1 WO 2023030534A1
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sequence
editing
seq
reverse transcriptase
pegrna
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French (fr)
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高彩霞
宗媛
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Suzhou QI Biodesign Biotechnology Co Ltd
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Suzhou QI Biodesign Biotechnology Co Ltd
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Priority to KR1020247010828A priority Critical patent/KR20240049838A/ko
Priority to US18/689,009 priority patent/US20240368609A1/en
Priority to JP2024514557A priority patent/JP2024532544A/ja
Priority to EP22863668.4A priority patent/EP4400594A4/en
Publication of WO2023030534A1 publication Critical patent/WO2023030534A1/zh
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Definitions

  • the invention relates to the field of genetic engineering.
  • the present invention relates to an improved guided editing system and a method for gene editing through the system.
  • genome editing technology such as CRISPR/Cas technology
  • the prime editing system is a system based on CRISPR/Cas technology that can precisely modify the sequence of the target site.
  • the system consists of two parts: 1) Cas9 nuclease (Cas9- H840A) and a fusion protein composed of reverse transcriptase (reverse transcriptase, RT); 2) a pegRNA with an RT template (RT template) and a free single-strand binding region (Primer binding site, PBS) at the 3' end.
  • the working principle of the system is to guide the fusion protein to bind to the designated site through the combination of PBS with the free single strand generated by Cas9-H840A, and reverse transcribe the single-stranded DNA sequence containing the specified mutation according to the given RT template. Repair, which can effect any change in the DNA sequence at a target site in the genome.
  • the present invention relates to by i) carrying out puncture to reverse transcriptase or deleting its superfluous structural domain such as RNase H structure domain; ii) reverse transcriptase and nucleocapsid protein NC fusion; Improve guide editing efficiency.
  • Embodiment 1 A guided editing system for targeted modification of plant genomes comprising:
  • a guide-editing fusion protein and/or an expression construct comprising a nucleotide sequence encoding said guide-editing fusion protein, wherein said guide-editing fusion protein comprises a CRISPR nickase and a reverse transcriptase;
  • At least one pegRNA and/or an expression construct comprising a nucleotide sequence encoding said at least one pegRNA
  • said at least one pegRNA comprises a guide sequence, a scaffold sequence, a reverse transcription (RT) template sequence and a primer binding site (PBS) sequence from 5' to 3' direction,
  • said at least one pegRNA is capable of forming a complex with said fusion protein and targeting said fusion protein to a target sequence in the genome, resulting in a nick within said target sequence.
  • Embodiment 2 The system of embodiment 1, wherein the CRISPR nickase is a Cas9 nickase, for example comprising the amino acid sequence shown in SEQ ID NO:2.
  • Embodiment 3 The system of embodiment 1 or 2, wherein the reverse transcriptase is M-MLV reverse transcriptase or a functional variant thereof.
  • Embodiment 4 The system of any one of embodiments 1-3, wherein the reverse transcriptase
  • connection sequence is missing;
  • Embodiment 5 The system of embodiment 4, wherein the reverse transcriptase comprises the sequence set forth in any one of SEQ ID NOs: 9-15.
  • Embodiment 6 The system of any one of embodiments 1-5, wherein in the fusion protein, the reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is linked to the nuclear Capsid protein (NC), hydrolase (PR) or integrase (IN) were fused directly or via a linker.
  • the reverse transcriptase such as M-MLV reverse transcriptase or a functional variant thereof
  • nucleocapsid protein comprises the amino acid sequence shown in SEQ ID NO:6
  • the hydrolase comprises the amino acid sequence shown in SEQ ID NO:7
  • Amino acid sequence or the integrase (IN) comprises the amino acid sequence shown in SEQ ID NO:8.
  • Embodiment 8 The system of embodiment 6 or 7, wherein the reverse transcriptase is fused to the nucleocapsid protein (NC) directly or through a joint at the N-terminus, preferably, the RNase H domain is deleted from the M- Functional variants of MLV reverse transcriptase are fused at the N-terminus to the nucleocapsid protein (NC), either directly or via a linker.
  • NC nucleocapsid protein
  • NC nucleocapsid protein
  • Embodiment 9 The system of any one of embodiments 1-8, wherein the guide sequence in the pegRNA is configured to have sufficient sequence identity to the target sequence to be capable of binding by base pairing to the complementary strand of the target sequence to achieve sequence specific targeting.
  • Embodiment 10 The system of any one of embodiments 1-9, wherein the scaffold sequence of the pegRNA comprises the sequence described in SEQ ID NO:17.
  • Embodiment 11 The system of any one of embodiments 1-10, wherein the primer binding sequence is configured to be complementary to at least a portion of the target sequence, preferably, the primer binding sequence is complementary to the 3 At least a part of the 'free single strand is complementary, especially complementary to the nucleotide sequence at the 3' end of the 3' free single strand.
  • Embodiment 12 The system of any one of embodiments 1-11, wherein the Tm (melting temperature) of the primer binding sequence is about 18°C to 52°C, preferably about 24°C to 36°C, more preferably about 28°C -32°C, more preferably about 30°C.
  • Embodiment 13 The system of any one of embodiments 1-12, wherein the RT template sequence is configured to correspond to a sequence downstream of the nick and to include the desired modification comprising one or more nucleotides substitutions, deletions and/or additions.
  • Embodiment 14 The system of any one of embodiments 1-13, wherein the guide editing fusion protein comprises the amino acid sequence set forth in SEQ ID NO:19.
  • a method of producing a genetically modified cell comprising introducing the guide editing system of any one of claims 1-14 into at least one of said cells, thereby resulting in modification of a target sequence in the genome of said at least one cell .
  • Figure 1 shows various modified PPE constructs.
  • Figure 2 shows the editing efficiencies of different constructs screened using the BFP-GFP reporter system in rice protoplasts obtained by flow cytometric analysis.
  • Figure 3 shows the guides obtained by amplicon sequencing analysis after the screened constructs PPE-NCv1, PPE-NCv2, and PPE- ⁇ RNase H with improved editing efficiency were co-transformed with pegRNA into rice protoplast cells. Editing efficiency of the editing system for different endogenous test loci.
  • Figure 4 shows PPE-NCv1, PPE- ⁇ RNase H, and the construct ePPE that was simultaneously fused with NC and deleted RNase H were co-transformed with pegRNA into rice protoplast cells, and analyzed by amplicon sequencing. Editing efficiency of the guided editing system for different endogenous test loci.
  • Figure 5 shows the expression level of PPE protein with improved editing efficiency in rice protoplasts.
  • Figure 6 shows that ePPE protein can effectively improve the editing efficiency of deletion or insertion of fragments larger than 15bp.
  • Figure 7 shows that ePPE proteins can effectively broaden the range of guided editing systems.
  • Figure 8 shows the editing efficiency of ePPE in rice callus.
  • Figure 9 shows ePPE creation of herbicide resistant rice plants.
  • Figure 10 shows editing of ePE in porcine cells.
  • Figure 11 shows that ePPE does not significantly increase the off-target editing efficiency in most endogenous test sites in rice, and the efficiency of the guided editing system obtained by amplicon sequencing analysis on different endogenous test sites and predicted endogenous off-target sites Editing efficiency.
  • Figure 12 shows the complementarity of ePPE and base editing at the edited site.
  • Figure 13 shows that the combination of ePPE with dual-pegRNA strategy and epegRNAs strategy further improves the efficiency of guide editing.
  • the term “and/or” covers all combinations of the items connected by the term, and each combination should be deemed to have been individually listed herein.
  • “A and/or B” includes “A,” “A and B,” and “B.”
  • “A, B, and/or C” encompasses “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”
  • the protein or nucleic acid may consist of said sequence, or may have additional amino acids or core elements at one or both ends of said protein or nucleic acid. Nucleotides, but still have the activity described in the present invention. In addition, those skilled in the art know that the methionine encoded by the start codon at the N-terminal of the polypeptide may be retained in some practical cases (such as when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide.
  • Gene as used herein encompasses not only chromosomal DNA present in the nucleus, but also organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).
  • organism includes any organism suitable for genome editing, preferably eukaryotes.
  • organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cows, cats; poultry such as chickens, ducks, geese; plants including monocots and dicots, Such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis, etc.
  • Genetically modified organism means an organism comprising within its genome an exogenous polynucleotide or comprising a modified gene or expression regulatory sequence.
  • exogenous polynucleotides can be stably integrated into the genome of an organism and inherited through successive generations.
  • Exogenous polynucleotides can be integrated into the genome either alone or as part of a recombinant DNA construct.
  • a modified gene or expression control sequence is one or more deoxynucleotide substitutions, deletions and additions to said gene or expression control sequence in the genome of an organism.
  • nucleic acid sequence is used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designations as follows: “A” for adenosine or deoxyadenosine (for RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or Deoxyguanosine, “U” means uridine, “T” means deoxythymidine, “R” means purine (A or G), “Y” means pyrimidine (C or T), “K” means G or T,” H” means A or C or T, “D” means A, T or G, “I” means inosine, and “N” means any nucleotide.
  • Polypeptide “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” may also include modified forms including, but not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation ylation and ADP-ribosylation.
  • expression construct refers to a vector such as a recombinant vector suitable for expressing a nucleotide sequence of interest in an organism.
  • “Expression” refers to the production of a functional product.
  • expression of a nucleotide sequence can refer to transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and/or translation of the RNA into a precursor or mature protein.
  • the "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be a translatable RNA (such as mRNA), such as RNA produced by in vitro transcription.
  • a translatable RNA such as mRNA
  • An "expression construct" of the present invention may comprise regulatory sequences and nucleotide sequences of interest of different origin, or regulatory sequences and nucleotide sequences of interest of the same origin but arranged in a manner different from that normally found in nature.
  • Promoter refers to a nucleic acid segment capable of controlling the transcription of another nucleic acid segment.
  • the promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from said cell.
  • the promoter may be a constitutive promoter or a tissue specific promoter or a developmentally regulated promoter or an inducible promoter.
  • promoters include, but are not limited to, polymerase (pol) I, pol II or pol III promoters.
  • pol I promoters include the chicken RNA pol I promoter.
  • pol II promoters include, but are not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the Simian virus 40 (SV40) immediate early promoter.
  • pol III promoters include the U6 and H1 promoters. Inducible promoters such as the metallothionein promoter can be used.
  • promoters include T7 phage promoter, T3 phage promoter, ⁇ -galactosidase promoter and Sp6 phage promoter.
  • the promoter can be cauliflower mosaic virus 35S promoter, corn Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, corn U3 promoter, rice actin promoter.
  • "Introducing" a nucleic acid molecule (eg, plasmid, linear nucleic acid fragment, RNA, etc.) or protein into an organism means transforming cells of the organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell.
  • "Transformation” as used in the present invention includes stable transformation and transient transformation.
  • “Stable transformation” refers to the introduction of exogenous nucleotide sequences into the genome, resulting in stable inheritance of exogenous genes. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.
  • Transient transformation refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of the foreign gene. In transient transformation, the exogenous nucleic acid sequence does not integrate into the genome.
  • Trait refers to a physiological, morphological, biochemical or physical characteristic of a cell or organism.
  • the present invention relates to a guided editing system for targeted modification of the genome DNA sequence of an organism, comprising:
  • a guide-editing fusion protein and/or an expression construct comprising a nucleotide sequence encoding said guide-editing fusion protein, wherein said guide-editing fusion protein comprises a CRISPR nickase and a reverse transcriptase;
  • At least one pegRNA and/or an expression construct comprising a nucleotide sequence encoding said at least one pegRNA
  • said at least one pegRNA comprises guide sequence, scaffold (scaffold) sequence, reverse transcription (RT) template sequence and primer binding site (PBS) sequence from 5' to 3' direction.
  • said CRISPR nickase and said reverse transcriptase in a direct editing fusion protein are linked by a linker.
  • the at least one pegRNA is capable of forming a complex with the fusion protein and targeting the fusion protein to a target sequence in the genome, resulting in a nick within the target sequence.
  • the invention relates to a guide editing fusion protein, wherein said guide editing fusion protein comprises a CRISPR nickase and a reverse transcriptase.
  • the present invention relates to the use of the guided editing fusion protein of the present invention in targeted modification of the genomic DNA sequence of an organism.
  • the organism is a plant.
  • guide editing system refers to the combination of components required for reverse transcription-based genome editing of the genome in a cell. Wherein the various components of the system, such as guide editing fusion protein, gRNA, etc., can exist independently, or can exist in any combination as a composition.
  • a "target sequence” refers to a sequence in the genome approximately 20 nucleotides in length characterized by 5' or 3' flanking PAM (prospacer adjacent motif) sequences.
  • the PAM is required for the recognition of the target sequence by the complex formed by the CRISPR nuclease or its variant and the guide RNA.
  • the target sequence is immediately adjacent to the PAM at the 3' end, such as 5'-NGG-3'. Based on the presence of PAMs, one skilled in the art can readily determine target sequences in the genome that are available for targeting. And depending on the location of the PAM, the target sequence can be located on either strand of the genomic DNA molecule.
  • the target sequence is preferably 20 nucleotides.
  • the CRISPR nickase in the fusion protein is capable of forming a nick within a target sequence in genomic DNA.
  • the CRISPR nickase is a Cas9 nickase.
  • the Cas9 nickase is derived from SpCas9 of S. pyogenes and comprises at least the amino acid substitution H840A relative to wild-type SpCas9.
  • Exemplary wild-type SpCas9 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the Cas9 nickase comprises the amino acid sequence shown in SEQ ID NO:2.
  • the Cas9 nickase in the fusion protein can be at the -3 nucleotide of the PAM of the target sequence (the first nucleotide at the 5' end of the PAM sequence is the +1 position) and the -4 core Nicks are formed between the nucleotides.
  • the Cas9 nickase is a Cas9 nickase variant capable of recognizing an altered PAM sequence. Many Cas9 nickase variants capable of recognizing altered PAM sequences are known in the art. In some embodiments, the Cas9 nickase is a Cas9 variant that recognizes the PAM sequence 5'-NG-3'.
  • the Cas9 nickase variant that recognizes the PAM sequence 5'-NG-3' comprises the following amino acid substitutions H840A, R1335V, L1111R, D1135V, G1218R, E1219F, A1322R, T1337R relative to wild-type Cas9, wherein the amino acid numbers are Refer to SEQ ID NO:1.
  • the nicking formed by the Cas9 nickase of the present invention can cause the target sequence to form a free single strand with a 3' end (3' free single strand) and a free single strand with a 5' end (5' free single strand).
  • the reverse transcriptases in the fusion proteins of the invention may be derived from different sources.
  • the reverse transcriptase is a reverse transcriptase of viral origin.
  • the reverse transcriptase is M-MLV reverse transcriptase or a functional variant thereof.
  • An exemplary wild-type M-MLV reverse transcriptase sequence is shown in SEQ ID NO:3.
  • the reverse transcriptase such as M-MLV reverse transcriptase or a functional variant thereof
  • connection sequence is missing;
  • the reverse transcriptase such as M-MLV reverse transcriptase or functional variant thereof comprises a mutation selected from D524N, and the amino acid position refers to SEQ ID NO:3.
  • the RNase H domain of said reverse transcriptase such as M-MLV reverse transcriptase or a functional variant thereof, is deleted.
  • connection sequence comprises the amino acid sequence shown in SEQ ID NO:4.
  • the RNase H domain comprises the amino acid sequence shown in SEQ ID NO:5.
  • the reverse transcriptase e.g., M-MLV reverse transcriptase or a functional variant thereof, comprises the sequence set forth in any one of SEQ ID NOs: 9-15.
  • the reverse transcriptase such as M-MLV reverse transcriptase or its functional variants is combined with nucleocapsid protein (NC), hydrolase (PR) at the N-terminal or C-terminal ) or integrase (IN) directly or through a linker fusion.
  • NC nucleocapsid protein
  • PR hydrolase
  • integrase integrase
  • the nucleocapsid protein (NC), hydrolase (PR) or integrase (IN) is for example from M-MLV.
  • the nucleocapsid protein comprises the amino acid sequence shown in SEQ ID NO:6.
  • the hydrolase (PR) comprises the amino acid sequence shown in SEQ ID NO:7.
  • the integrase (IN) comprises the amino acid sequence shown in SEQ ID NO:8.
  • the reverse transcriptase such as M-MLV reverse transcriptase or a functional variant thereof is fused at the N-terminus to the nucleocapsid protein (NC) directly or via a linker.
  • the reverse transcriptase such as M-MLV reverse transcriptase or a functional variant thereof is fused at the C-terminus to the nucleocapsid protein (NC) directly or via a linker.
  • a “linker” can be 1-50 in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20-25, 25-50) or more amino acids, non-functional amino acid sequences without secondary structures.
  • the linker can be a flexible linker such as GGGGS, GS, GAP, (GGGGS)x3, GGS, and (GGS)x7, etc.
  • it can be the linker shown in SEQ ID NO: 16.
  • the CRISPR nickase in the fusion protein is N-terminal to the reverse transcriptase. In some embodiments, the CRISPR nickase in the fusion protein is located C-terminal to the reverse transcriptase.
  • the fusion protein of the present invention may further comprise a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • the one or more NLSs in the fusion protein should be of sufficient strength to drive accumulation of the fusion protein in the nucleus of the cell in an amount that enables its base editing function.
  • the strength of nuclear localization activity is determined by the number of NLSs in the fusion protein, their location, the particular NLS(s) used, or a combination of these factors.
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the guide sequence (also known as seed sequence or spacer sequence) in at least one pegRNA of the present invention is set to have sufficient sequence identity (preferably 100% identity) with the target sequence, thereby can be with the target sequence through base pairing Complementary strand binding for sequence-specific targeting.
  • scaffold sequences for gRNAs suitable for genome editing based on CRISPR nucleases are known in the art, and these can be used in the pegRNAs of the present invention.
  • the scaffold sequence of the gRNA is shown in SEQ ID NO: 17.
  • the primer binding sequence is configured to be complementary to at least a part of the target sequence (preferably completely paired with at least a part of the target sequence), preferably, the primer binding sequence is compatible with the DNA where the target sequence is located. At least a part of the 3' free single strand caused by the nicking in the chain is complementary (preferably completely paired with at least a part of the 3' free single strand), especially a nucleotide sequence with the 3' end of the 3' free single strand Complementary (preferably perfect pairing).
  • the 3' free single strand of the chain When the 3' free single strand of the chain is combined with the primer binding sequence through base pairing, the 3' free single strand can be used as a primer to form a reverse transcription (RT) template immediately adjacent to the primer binding sequence
  • RT reverse transcription
  • the sequence is used as a template, reverse-transcribed under the action of reverse transcriptase in the fusion protein, and a DNA sequence corresponding to the reverse-transcribed (RT) template sequence is extended.
  • the primer binding sequence depends on the length of the free single strand formed in the target sequence by the CRISPR nickase used, however, it should be of the minimum length to ensure specific binding.
  • the length of the primer binding sequence can be 4-20 nucleotides, for example, the length is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 nucleotides.
  • the primer binding sequence is configured to have a Tm (melting temperature) of no more than about 52°C. In some embodiments, the Tm (melting temperature) of the primer binding sequence is about 18°C-52°C, preferably about 24°C-36°C, more preferably about 28°C-32°C, more preferably about 30°C.
  • the method for calculating the Tm of a nucleic acid sequence is well known in the art, for example, it can be calculated using an online analysis tool of Oligo Analysis Tool.
  • a suitable Tm can be obtained by selecting a suitable length of PBS.
  • a PBS sequence with an appropriate Tm can be obtained by selecting an appropriate target sequence.
  • the RT template sequence can be any sequence. Through the above reverse transcription, its sequence information can be integrated into the DNA strand where the target sequence is located (that is, the strand containing the target sequence PAM), and then through the DNA repair function of the cell, a DNA double strand containing the sequence information of the RT template is formed .
  • the RT template sequence comprises desired modifications.
  • the desired modification includes substitution, deletion and/or addition of one or more nucleotides.
  • the modification includes one or more substitutions selected from: C to T substitution, C to G substitution, C to A substitution, G to T substitution, G to C substitution, G to A substitution, A to T substitution , A to G substitution, A to C substitution, T to C substitution, T to G substitution, T to A substitution; and/or a deletion comprising one or more nucleotides, e.g., 1 to about 100 or more 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions and/or include insertions of one or more nucleotides, for example 1 to about 100 or more, for example 1 to about 100 or more, for example 1, 2, 3, 4 Insertion of about 1, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotides.
  • the RT template sequence is configured to correspond to (e.g., be complementary to at least a portion of) the sequence downstream of the target sequence nick, and to comprise the desired modification.
  • desired modifications include substitutions, deletions and/or additions of one or more nucleotides.
  • the modification includes one or more substitutions selected from: C to T substitution, C to G substitution, C to A substitution, G to T substitution, G to C substitution, G to A substitution, A to T substitution , A to G substitution, A to C substitution, T to C substitution, T to G substitution, T to A substitution; and/or a deletion comprising one or more nucleotides, e.g., 1 to about 100 or more 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions and/or include insertions of one or more nucleotides, for example 1 to about 100 or more, for example 1 to about 100 or more, for example 1, 2, 3, 4 Insertion of about 1, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotides.
  • the RT template sequence can be about 1-300 or more nucleotides in length, for example, 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275 , a polynucleotide of about 300 nucleotides or more.
  • the length of the RT template sequence is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 nucleotides.
  • the guide editing system further includes a nicking gRNA (nicking gRNA, used to generate an additional nick) and/or an expression construct comprising a nucleotide sequence encoding the nicking gRNA, the nicking gRNA comprising a guide Sequence and scaffold sequence.
  • the nicked gRNA does not comprise a reverse transcription (RT) template sequence and a primer binding site (PBS) sequence.
  • the guide sequence (also known as seed sequence or spacer sequence) in the nicking gRNA of the present invention is set to have sufficient sequence identity (preferably 100% identity) with the nicking target sequence in the genome, thereby being able to target the fusion protein of the present invention
  • the nicking target sequence is located on the opposite strand of the genomic DNA from the target sequence targeted by the pegRNA (pegRNA target sequence) and results in a nick within the nicking target sequence.
  • the nick formed by the nicking RNA and the nick formed by the pegRNA are separated by about 1 to about 300 or more nucleotides, such as 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225 , about 250, about 275, about 300 nucleotides or more.
  • the nicking formed by the nicking RNA is located upstream or downstream of the formation of the pegRNA (both upstream and downstream refer to the DNA strand where the pegRNA target sequence is located).
  • the guide sequence in the nicking gRNA has sufficient sequence identity (preferably 100% identity) to the opposite strand (modified) of the pegRNA target sequence after the editing event, so that the nicking gRNA only targets To the nicked target sequence that occurs after pegRNA-induced targeting and modification of the target sequence is complete.
  • the PAM of the nicking target sequence is located within the complement of the pegRNA target sequence.
  • the pegRNA and/or nicked gRNA can be precisely processed to its sequence using a self-processing system.
  • the 5' end of the pegRNA and/or nicking gRNA is connected to the 3' end of a first ribozyme, and the first ribozyme is designed to be at the 5' end of the pegRNA and/or nicking gRNA. cleave the fusion at the ' end; and/or the 3' end of the pegRNA and/or nicked gRNA is linked to the 5' end of a second ribozyme designed to be present in the pegRNA and/or The 3' end of the nick gRNA cleaves the fusion.
  • first or second ribozyme is within the purview of those skilled in the art. For example, see Gao et al., JIPB, Apr, 2014; Vol 56, Issue 4, 343-349. Methods for precise processing of gRNA can be found, for example, in WO 2018/149418.
  • the guide editing system comprises at least one pair of pegRNAs and/or an expression construct comprising a nucleotide sequence encoding the at least one pair of pegRNAs.
  • the two pegRNAs of the pegRNA pair are configured to target different target sequences on the same strand of genomic DNA.
  • the two pegRNAs of the pegRNA pair are configured to target target sequences on different strands of genomic DNA.
  • the PAM of the target sequence of one pegRNA of the pegRNA pair is on the sense strand and the PAM of the other pegRNA is on the antisense strand.
  • the induced nicks of the two pegRNAs are respectively located on both sides of the site to be modified.
  • the pegRNA-induced nicking for the sense strand is upstream (5' direction) of the site to be modified, and the pegRNA-induced nicking for the antisense strand is downstream (3' direction) of the site to be modified. Said upstream or downstream is with respect to the sense strand.
  • the induced nicks of the two pegRNAs are about 1 to about 300 or more nucleotides apart, eg, 1-15 nucleotides apart.
  • both pegRNAs of the pegRNA pair are configured to introduce the same desired modification.
  • one of the pegRNAs is configured to introduce substitutions from A to G in the sense strand, while the other pegRNA is configured to introduce substitutions from T to C in the corresponding position of the antisense strand.
  • one of the pegRNAs is configured to introduce a two-nucleotide deletion in the sense strand, and the other pegRNA is configured to also introduce a two-nucleotide deletion in the corresponding position of the antisense strand.
  • Other types of modification can be deduced by analogy.
  • PegRNAs targeting two different strands, respectively, can achieve the same desired modification by designing appropriate RT template sequences.
  • the pegRNA of the present invention is epegRNA.
  • the construction of epegRNA can refer to James W.Nelson et al., Engineered pegRNAs improve prime editing efficiency. Nature Biotechnology volume 40, 402-410 (2022), which is incorporated herein by reference.
  • the epegRNA is an epegRNA modified with a 3'-tevopreQ1-8nt linker.
  • the nucleotide sequence encoding the fusion protein is codon-optimized for the organism species whose genome is to be modified.
  • Codon optimization refers to the replacement of at least one codon of a native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10 , 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence and modifying the nucleic acid sequence in order to enhance expression in the host cell of interest.
  • a native sequence e.g., about or more than about 1, 2, 3, 4, 5, 10 , 15, 20, 25, 50 or more codons
  • Codon bias differences in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon Usage Database Codon Usage Database
  • Codon Usage Database available at www.kazusa.orjp/codon/
  • these tables can be adjusted in different ways Applicable. See, Nakamura Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).
  • the invention provides a method of producing a genetically modified cell comprising introducing into at least one of said cells a guide editing system of the invention, thereby resulting in modification of a target sequence in the genome of said at least one cell.
  • the modifications include substitutions, deletions and/or additions of one or more nucleotides.
  • the modification includes one or more substitutions selected from: C to T substitution, C to G substitution, C to A substitution, G to T substitution, G to C substitution, G to A substitution, A to T substitution , A to G substitution, A to C substitution, T to C substitution, T to G substitution, T to A substitution; and/or a deletion comprising one or more nucleotides, e.g., 1 to about 100 or more 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions and/or include insertions of one or more nucleotides, for example 1 to about 100 or more, for example 1 to about 100 or more, for example 1, 2, 3, 4 Insertions of 1, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotides
  • the present invention also provides a method for producing a genetically modified cell, comprising introducing the guide editing system of the present invention into the cell.
  • the invention also provides a genetically modified organism comprising a genetically modified cell produced by a method of the invention, or a progeny thereof.
  • the target sequence to be modified can be located anywhere in the genome, for example, in a functional gene such as a protein-coding gene, or for example, in a gene expression regulatory region such as a promoter region or an enhancer region, so as to realize the Modification of gene function or modification of gene expression. Modifications in the cellular target sequence can be detected by T7EI, PCR/RE or sequencing methods.
  • the guide editing system can be introduced into cells by various methods well known to those skilled in the art.
  • Methods that can be used to introduce the guide editing system of the present invention into cells include, but are not limited to: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, etc. virus, adeno-associated virus, lentivirus, and other viruses), particle bombardment, PEG-mediated transformation of protoplasts, Agrobacterium-mediated transformation.
  • Cells that can be gene-edited by the method of the present invention can be derived from, for example, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; plants, including monads Leafy and dicotyledonous plants, such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis, etc.
  • the methods of the invention are performed in vitro.
  • the cell is an isolated cell, or a cell in an isolated tissue or organ.
  • the methods of the invention can also be performed in vivo.
  • the cell is a cell in an organism into which the system of the present invention can be introduced in vivo by, for example, a virus- or Agrobacterium-mediated method.
  • the invention provides a method of producing a genetically modified plant comprising introducing into at least one of said plants a guide editing system of the invention, thereby resulting in a modification in the genome of said at least one plant.
  • the modifications include substitutions, deletions and/or additions of one or more nucleotides.
  • the modification includes one or more substitutions selected from: C to T substitution, C to G substitution, C to A substitution, G to T substitution, G to C substitution, G to A substitution, A to T substitution , A to G substitution, A to C substitution, T to C substitution, T to G substitution, T to A substitution; and/or a deletion comprising one or more nucleotides, e.g., 1 to about 100 or more 1, 2, 3, 4, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotide deletions and/or include insertions of one or more nucleotides, for example 1 to about 100 or more, for example 1 to about 100 or more, for example 1, 2, 3, 4 Insertion of about 1, 5, about 10, about 20, about 30, about 40, about 50, about 75, about 100 nucleotides.
  • the method further comprises selecting from said at least one plant for plants having the desired modification.
  • the guide editing system can be introduced into plants by various methods well known to those skilled in the art.
  • Methods that can be used to introduce the guide editing system of the present invention into plants include, but are not limited to: biolistic method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube passage method, and ovary injection method.
  • said genome editing system is introduced into plants by transient transformation.
  • the modification of the genome can be realized only by introducing or producing the guide editing fusion protein and gRNA in the plant cell, and the modification can be stably inherited without the need to encode the components of the editing system Stably transformed plants with exogenous polynucleotides. In this way, the potential off-target effect of the stably existing (continuously produced) editing system is avoided, and the integration of exogenous nucleotide sequences in the plant genome is avoided, thereby having higher biological safety.
  • the introduction is performed in the absence of selection pressure, thereby avoiding the integration of the exogenous nucleotide sequence in the plant genome.
  • the introducing comprises transforming an isolated plant cell or tissue with a guide editing system of the invention, and then regenerating the transformed plant cell or tissue into a whole plant.
  • said regeneration is performed in the absence of selection pressure, ie without using any selection agent for the selection gene carried on the expression vector during tissue culture.
  • the regeneration efficiency of plants can be improved without using a selection agent, and modified plants without foreign nucleotide sequences can be obtained.
  • the guide editing system of the present invention can be transformed into specific parts on a whole plant, such as leaves, shoot tips, pollen tubes, young ears or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate in tissue culture.
  • the in vitro expressed protein and/or the in vitro transcribed RNA molecule (eg, the expression construct is an in vitro transcribed RNA molecule) is transformed directly into the plant.
  • the protein and/or RNA molecule can realize genome editing in plant cells, and then be degraded by cells, avoiding the integration of exogenous nucleotide sequences in plant genomes.
  • genetically modifying and breeding plants using the method of the present invention can obtain plants whose genomes have no integration of exogenous polynucleotides, that is, non-transgene-free modified plants.
  • the method further comprises culturing the plant cell, tissue or whole plant into which the guide editing system has been introduced at an elevated temperature, such as 37°C.
  • said modified genomic region is associated with a plant trait such as an agronomic trait, whereby said modified substitution results in said plant having an altered (preferably improved) trait relative to a wild-type plant, For example agronomic traits.
  • the method further comprises the step of screening for plants having a desired modification and/or a desired trait, such as an agronomic trait.
  • the method further comprises obtaining progeny of the genetically modified plant.
  • the genetically modified plant or progeny thereof has the desired modification and/or a desired trait such as an agronomic trait.
  • the present invention also provides a genetically modified plant or progeny or part thereof, wherein said plant is obtained by the above-mentioned method of the present invention.
  • the genetically modified plant or progeny or part thereof is non-transgenic.
  • said genetically modified plant or progeny thereof has the desired genetic modification and/or desired traits such as agronomic traits.
  • the present invention also provides a method of plant breeding, comprising crossing a genetically modified first plant obtained by the above-mentioned method of the present invention with a second plant that does not contain the modification, so that the modified Import the second plant.
  • said first genetically modified plant has a desired trait, such as an agronomic trait.
  • Agronomic traits specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf greenness, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit Yield, seed yield, plant total nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, plant total free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, total plant protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, herbicide resistance and drought resistance, nitrogen absorption, root lodging, harvest index, stem lodging, plant height, ear height, ear length, disease resistance Sex, cold resistance, salt resistance and tiller number, etc.
  • the present invention also includes a kit used in the method of the present invention, the kit comprising at least the guide-editing fusion protein of the present invention or an expression construct of the guide-editing fusion protein.
  • the kit can also include expression constructs and related reagents for preparing pegRNA.
  • the kit may also comprise reagents for introducing the guide editing system into an organism or cells of an organism.
  • Kits generally include a label indicating the intended use and/or method of use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the gene fragment encoding PPE (SEQ ID NO: 18) was subjected to point mutation or deletion of the corresponding fragment at the corresponding site of M-MLV reverse transcriptase by PCR to obtain encoding PPE-F155Y, PPE-F155V, PPE-F156Y, PPE respectively -D524N, PPE-N200C, PPE- ⁇ RNase H, PPE- ⁇ RNase H- ⁇ Connection gene fragments were connected by Gibson method to obtain the corresponding final vector.
  • the M-MLV reverse transcriptase sequences comprising F155Y, F155V, F156Y, D524N, and N200C are shown in SEQ ID NO: 9-13, respectively.
  • the sequence of M-MLV reverse transcriptase ⁇ RNase H is shown in SEQ ID NO:14.
  • the sequence of M-MLV reverse transcriptase ⁇ RNase H- ⁇ Connection is shown in SEQ ID NO:15.
  • nucleocapsid protein NC SEQ ID NO: 6
  • hydrolase PR SEQ ID NO: 7
  • integrase IN SEQ ID NO: 8
  • nucleocapsid protein NC was fused to the 5' end of the M-MLV reverse transcriptase ⁇ RNase H gene fragment encoding PPE- ⁇ RNase H after plant codon optimization to obtain PPE- ⁇ RNase H-NC (enhanced PPE, ePPE, the amino acid sequence is shown in SEQ ID NO: 19), and then constructed by the Gibson method to obtain the corresponding final vector.
  • the specific carrier is shown in Figure 1.
  • the pegRNA fragment (including RT template and PBS sequence) was constructed on the vector driven by the OsU3 promoter using the Gibson method to obtain the OsU3-pegRNA construct suitable for rice.
  • the protoplasts used in the present invention come from the rice variety Zhonghua 11.
  • the rice variety is Zhonghua 11, and the seeds are first rinsed with 75% ethanol for 1 minute, then treated with 4% sodium hypochlorite for 30 minutes, and washed with sterile water for more than 5 times. Placed on M6 medium for 3-4 weeks, 26 ° C, protected from light.
  • Protoplasts were collected in 2mL centrifuge tubes, protoplast DNA ( ⁇ 30 ⁇ L) was extracted by CTAB method, and its concentration (30-60ng/ ⁇ L) was measured by NanoDrop ultramicro spectrophotometer, and stored at -20°C.
  • the protoplast DNA template was amplified by PCR using genomic primers.
  • 20 ⁇ L amplification system contains 4 ⁇ L 5 ⁇ Fastpfu buffer, 1.6 ⁇ L dNTPs (2.5mM), 0.4 ⁇ L Forward primer (10 ⁇ M), 0.4 ⁇ L Reverse primer (10 ⁇ M), 0.4 ⁇ L FastPfu polymerase (2.5U/ ⁇ L), and 2 ⁇ L DNA template ( ⁇ 60ng).
  • Amplification conditions pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 50-64°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles; full extension at 72°C for 5 minutes, storage at 12°C;
  • the above-mentioned amplification products were diluted 10 times, and 1 ⁇ L was used as the template for the second round of PCR amplification, and the amplification primers were sequencing primers containing Barcode.
  • the 50 ⁇ L amplification system contains 10 ⁇ L 5 ⁇ Fastpfu buffer, 4 ⁇ L dNTPs (2.5mM), 1 ⁇ L Forward primer (10 ⁇ M), 1 ⁇ L Reverse primer (10 ⁇ M), 1 ⁇ L FastPfu polymerase (2.5U/ ⁇ L), and 1 ⁇ L DNA template.
  • the amplification conditions were as above, and the number of amplification cycles was 35 cycles.
  • PCR products were separated by electrophoresis on 2% agarose gel, and the target fragments were gel-recovered using the AxyPrep DNA Gel Extraction kit, and the recovered products were quantitatively analyzed using a NanoDrop ultra-micro spectrophotometer; 100ng of the recovered products were mixed respectively, And sent to Sangon Bioengineering Co., Ltd. for amplicon sequencing library construction and amplicon sequencing analysis.
  • Example 1 Screening of different constructs using a reporter system
  • the BFP-GFP reporter system was used for preliminary screening in rice protoplasts. Through flow cytometry analysis, the results are shown in Figure 2, indicating that the editing efficiency of PPE- ⁇ RNase H, PPE-NCv1 and PPE-NCv2 has improved compared with the original PPE. These three constructs were therefore selected for subsequent endogenous target testing.
  • Example 3 The combination of different PPE protein modifications can further improve the editing efficiency of the guided editing system
  • the corresponding protein was transformed into rice protoplasts, and the protoplast proteins were extracted and then Western-Blot was performed.
  • the results are shown in the figure 5, it shows that the expression levels of PPE-NCv1, PPE-NCv2, PPE- ⁇ RNase H, and ePPE proteins are significantly higher than that of the original PPE protein, and the protein expression level of ePPE is the highest, corresponding to the level of its guided editing efficiency.
  • Example 5 ePPE protein can effectively improve the editing efficiency of fragment deletion or insertion greater than 15bp
  • Example 6 ePPE protein can effectively broaden the scope of the guided editing system
  • Example 7 Editing efficiency of ePPE in rice callus
  • Example 8 ePPE creates herbicide-resistant rice plants
  • ePE In order to test the editing efficiency of ePPE in other species, three targets of porcine cells were selected for testing (named ePE). The results are shown in Figure 10. Compared with the original PE treatment, ePE does have different degrees of efficiency improvement.
  • Example 12 The combination of ePPE and pegRNA optimization strategy further improves the efficiency of guided editing
  • the ePPE protein was combined with the dual-pegRNA strategy and the epegRNAs strategy.
  • the results are shown in Figure 13.
  • the final choice was to construct a dual-pegRNA modified with 3'-tevopreQ1-8nt linker to transform protoplasts together with PPE and ePPE, and to combine the dual-pegRNA strategy and epegRNAs strategy for guided editing
  • the efficiency was further improved, and the combination of ePPE-dual-epegRNA was 7.9 times higher than that of PPE-dual-pegRNA.

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Abstract

提供了一种改进的引导编辑系统，以及通过该系统进行基因编辑的方法。

Description

改进的引导编辑系统 技术领域
本发明涉及基因工程领域。具体而言，本发明涉及一种改进的引导编辑系统，以及通过该系统进行基因编辑的方法。
发明背景
许多重要的疾病和农艺性状都决定于基因组的序列。通过对基因组特定序列进行定向的改变，能够赋予生物体新的可遗传的性状，从而为疾病治疗和育种改良提供可能。目前，通过基因组编辑技术(例如CRISPR/Cas技术)可以实现对特定序列进行编辑，进而激活细胞的修复途径对损伤处进行修复。引导编辑系统(Prime Editing，PE)是一种基于CRISPR/Cas技术的可以精确修改靶位点序列的系统，该系统由两部分构成：1)具有非靶标链缺刻活性的Cas9核酸酶(Cas9-H840A)和逆转录酶(reverse transcriptase，RT)构成的融合蛋白；2)3’端带有RT模板(RT template)和游离单链的结合区(Primer binding site，PBS)的pegRNA。该系统的工作原理是通过PBS结合Cas9-H840A所产生的游离单链，引导融合蛋白结合至指定位点，并依照给定的RT模板逆转录出含有指定突变的单链DNA序列，经过细胞的修复，可以在基因组中实现位于靶位点处的DNA序列的任意变化。
目前研究表明，PE编辑效率整体较低，需要进行多方面的优化。通过改造M-MLV蛋白或者融合其他相关蛋白从而增加PE蛋白的稳定性或表达量是一种提高PE编辑效率的有效途径。
发明简述
本发明涉及通过i)对逆转录酶进行点突或者删除其多余结构域如RNase H结构域；ii)将逆转录酶与核衣壳蛋白NC融合；或i)和ii)两者的组合来提高引导编辑效率。
本发明至少包含以下实施方案：
实施方案1.一种用于靶向性修饰植物基因组的引导编辑系统，其包含：
i)引导编辑融合蛋白和/或含有编码所述引导编辑融合蛋白的核苷酸序列的表达构建体，其中所述引导编辑融合蛋白包含CRISPR切口酶和逆转录酶；和/或
ii)至少一种pegRNA和/或含有编码所述至少一种pegRNA的核苷酸序列的表达构建体，
其中所述至少一种pegRNA从5’至3’方向包含引导序列、支架序列、反转录(RT)模板序列和引物结合位点(PBS)序列，
其中所述至少一种pegRNA能够与所述融合蛋白形成复合物并将所述融合蛋白靶 向基因组中的靶序列，导致所述靶序列内的切口。
实施方案2.实施方案1的系统，其中所述CRISPR切口酶是Cas9切口酶，例如包含SEQ ID NO:2所示氨基酸序列。
实施方案3.实施方案1或2的系统，其中所述逆转录酶是M-MLV逆转录酶或其功能性变体。
实施方案4.实施方案1-3中任一项的系统，其中所述逆转录酶
(a)包含选自F155Y、F155V、F156Y、D524N、N200C中任一种的突变，所述氨基酸位置参考SEQ ID NO:3；
(b)connection序列被缺失；和/或
(c)RNase H结构域被突变或缺失。
实施方案5.实施方案4的系统，其中所述逆转录酶包含SEQ ID NO:9-15中任一项所述的序列。
实施方案6.实施方案1-5中任一项的系统，其中在所述融合蛋白中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体在N端或C端与核衣壳蛋白(NC)、水解酶(PR)或整合酶(IN)直接地或通过接头融合。
实施方案7.实施方案6的系统，所述核衣壳蛋白(NC)包含如SEQ ID NO:6所示的氨基酸序列，或所述水解酶(PR)包含如SEQ ID NO:7所示的氨基酸序列，或所述整合酶(IN)包含如SEQ ID NO:8所示的氨基酸序列。
实施方案8.实施方案6或7的系统，其中所述逆转录酶在N端与核衣壳蛋白(NC)直接地或通过接头融合，优选地，所述RNase H结构域被缺失的M-MLV逆转录酶的功能性变体在N端与核衣壳蛋白(NC)直接地或通过接头融合。
实施方案9.实施方案1-8中任一项的系统，其中pegRNA中的引导序列被设置为与靶序列具有充分序列相同性，从而能够通过碱基配对与靶序列的互补链结合，实现序列特异性靶向。
实施方案10.实施方案1-9中任一项的系统，其中所述pegRNA的支架序列包含SEQ ID NO:17所述序列。
实施方案11.实施方案1-10中任一项的系统，其中所述引物结合序列被设置为与所述靶序列的至少一部分互补，优选地，所述引物结合序列与所述切口导致的3’游离单链的至少一部分互补，特别是与所述3’游离单链的3’末端的核苷酸序列互补。
实施方案12.实施方案1-11中任一项的系统，其中所述引物结合序列的Tm(解链温度)为大约18℃-52℃，优选大约24℃-36℃，更优选大约28℃-32℃，更优选大约30℃。
实施方案13.实施方案1-12中任一项的系统，其中，所述RT模板序列被设置为对应于切口下游的序列，并包含期望的修饰，所述修饰包括一或多个核苷酸的取代、缺失和/或添加。
实施方案14.实施方案1-13中任一项的系统，其中所述引导编辑融合蛋白包含SEQ ID NO:19所示氨基酸序列。
15.一种产生经遗传修饰的细胞的方法，包括将权利要求1-14中任一项的引导编辑系统导入至少一个所述细胞，由此导致所述至少一个细胞的基因组中靶序列的修饰。
附图简述
图1示出了经修饰的不同PPE构建体。
图2示出了通过流式细胞分析获得的利用BFP-GFP报告系统筛选的不同构建体在水稻原生质体中的编辑效率。
图3示出了将筛选出的具有提高的编辑效率的构建体PPE-NCv1、PPE-NCv2和PPE-ΔRNase H与pegRNA共转化至水稻原生质体细胞中后，通过扩增子测序分析得到的引导编辑系统对不同内源测试位点的编辑效率。
图4示出了将PPE-NCv1、PPE-ΔRNase H和同时融合NC和删除RNase H的组合处理的构建体ePPE分别与pegRNA共转化至水稻原生质体细胞中后，通过扩增子测序分析得到的引导编辑系统对不同内源测试位点的编辑效率。
图5示出了具有提高的编辑效率的PPE蛋白在水稻原生质体中的表达量。
图6示出ePPE蛋白可有效提高大于15bp片段删除或插入的编辑效率。
图7示出ePPE蛋白可有效拓宽引导编辑系统范围。
图8示出ePPE在水稻愈伤中的编辑效率。
图9示出ePPE创制抗除草剂水稻植物。
图10示出ePE在猪细胞中的编辑。
图11示出ePPE在大多数水稻内源测试位点中不会显著增加脱靶编辑效率，通过扩增子测序分析得到的引导编辑系统对不同内源测试位点及预测的内源脱靶位点的编辑效率。
图12示出ePPE与碱基编辑在编辑位点实现功能的互补。
图13示出ePPE结合dual-pegRNA strategy策略、epegRNAs策略相结合进一步提高引导编辑效率。
发明详述
一、定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989(下文称为“Sambrook”)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达系统表达时)会被保留，但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的多肽氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸的序列，相应地，其编码核苷酸序列也可以包含起始密码子；反之亦然。
“基因组”如本文所用不仅涵盖存在于细胞核中的染色体DNA，而且还包括存在于细胞的亚细胞组分(如线粒体、质体)中的细胞器DNA。
如本文所用，“生物体”包括适于基因组编辑的任何生物体，优选真核生物。生物体的实例包括但不限于，哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。
“经遗传修饰的生物体”意指在其基因组内包含外源多核苷酸或包含经修饰的基因或表达调控序列的生物体。例如外源多核苷酸能够稳定地整合进生物体的基因组中，并遗传连续的世代。外源多核苷酸可单独地或作为重组DNA构建体的部分整合进基因组中。经修饰的基因或表达调控序列为在生物体基因组中所述基因或表达调控序列包含一个或多个脱氧核苷酸取代、缺失和添加。
“多核苷酸”、“核酸序列”、“核苷酸序列”或“核酸片段”可互换使用并且是单链或双链RNA或DNA聚合物，任选地可含有合成的、非天然的或改变的核苷酸碱基。核苷酸通过如下它们的单个字母名称来指代：“A”为腺苷或脱氧腺苷(分别对应RNA或DNA)，“C”表示胞苷或脱氧胞苷，“G”表示鸟苷或脱氧鸟苷，“U”表示尿苷，“T”表示脱氧胸苷，“R”表示嘌呤(A或G)，“Y”表示嘧啶(C或T)，“K”表示G或T，“H”表示A或C或T，“D”表示A、T或G，“I”表示肌苷，并且“N”表示任何核苷酸。
“多肽”、“肽”、和“蛋白”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列 的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。
本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，在一些实施方式中，可以是能够翻译的RNA(如mRNA)，例如是体外转录生成的RNA。
本发明的“表达构建体”可包含不同来源的调控序列和感兴趣的核苷酸序列，或相同来源但以不同于通常天然存在的方式排列的调控序列和感兴趣的核苷酸序列。
“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方式中，启动子是能够控制细胞中基因转录的启动子，无论其是否来源于所述细胞。启动子可以是组成型启动子或组织特异性启动子或发育调控启动子或诱导型启动子。
启动子的实例包括但不限于聚合酶(pol)I、pol II或pol III启动子。pol I启动子的实例包括鸡RNA pol I启动子。pol II启动子的实例包括但不限于巨细胞病毒立即早期(CMV)启动子、劳斯肉瘤病毒长末端重复(RSV-LTR)启动子和猿猴病毒40(SV40)立即早期启动子。pol III启动子的实例包括U6和H1启动子。可以使用诱导型启动子如金属硫蛋白启动子。启动子的其他实例包括T7噬菌体启动子、T3噬菌体启动子、β-半乳糖苷酶启动子和Sp6噬菌体启动子。当用于植物时，启动子可以是花椰菜花叶病毒35S启动子、玉米Ubi-1启动子、小麦U6启动子、水稻U3启动子、玉米U3启动子、水稻肌动蛋白启动子。
将核酸分子(例如质粒、线性核酸片段、RNA等)或蛋白质“导入”生物体是指用所述核酸或蛋白质转化生物体细胞，使得所述核酸或蛋白质在细胞中能够发挥功能。本发明所用的“转化”包括稳定转化和瞬时转化。“稳定转化”指将外源核苷酸序列导入基因组中，导致外源基因稳定遗传。一旦稳定转化，外源核酸序列稳定地整合进所述生物体和其任何连续世代的基因组中。“瞬时转化”指将核酸分子或蛋白质导入细胞中，执行功能而没有外源基因稳定遗传。瞬时转化中，外源核酸序列不整合进基因组中。
“性状”指细胞或生物体的生理的、形态的、生化的或物理的特征。
二、改进的引导编辑系统
在一方面，本发明涉及一种用于靶向性修饰生物体基因组DNA序列的引导编辑系统，其包含：
i)引导编辑融合蛋白和/或含有编码所述引导编辑融合蛋白的核苷酸序列的表达构建体，其中所述引导编辑融合蛋白包含CRISPR切口酶和逆转录酶；和/或
ii)至少一种pegRNA和/或含有编码所述至少一种pegRNA的核苷酸序列的表达构建体，
其中所述至少一种pegRNA从5’至3’方向包含引导序列、支架(scaffold)序列、逆转录(RT)模板序列和引物结合位点(PBS)序列。
在一些实施方案中，引导编辑融合蛋白中的所述CRISPR切口酶和所述逆转录酶通过接头相连。
在一些实施方案中，所述至少一种pegRNA能够与所述融合蛋白形成复合物并将所 述融合蛋白靶向基因组中的靶序列，导致所述靶序列内的切口。
在一方面，本发明涉及一种引导编辑融合蛋白，其中所述引导编辑融合蛋白包含CRISPR切口酶和逆转录酶。
在一方面，本发明涉及本发明的引导编辑融合蛋白在靶向性修饰生物体基因组DNA序列中的用途。
在一些实施方案中，所述生物体是植物。
如本文所用，“引导编辑系统”是指用于对细胞内基因组进行基于逆转录的基因组编辑所需的成分的组合。其中所述系统的各个成分，例如引导编辑融合蛋白、gRNA等可以各自独立地存在，或者可以以任意的组合作为组合物的形式存在。
如本文所用，“靶序列”是指基因组中由5’或3’侧翼的PAM(前间区序列邻近基序)序列所表征的长度大约20个核苷酸的序列。通常而言，PAM是CRISPR核酸酶或其变体与向导RNA形成的复合物识别靶序列所必需的。例如，对于Cas9核酸酶及其变体，其靶序列在3’末端紧邻PAM，例如5’-NGG-3’。基于PAM的存在，本领域技术人员可以容易地确定基因组中可用于靶向的靶序列。而且取决于PAM的位置，靶序列可以位于基因组DNA分子的任一条链上。对于Cas9或其衍生物例如Cas9切口酶而言，靶序列优选20个核苷酸。
在一些实施方案中，融合蛋白中的所述CRISPR切口酶(nickase)能够在基因组DNA中靶序列内形成切口(nick)。在一些实施方案中，所述CRISPR切口酶是Cas9切口酶。
在一些实施方案中，所述Cas9切口酶衍生自化脓链球菌(S.pyogenes)的SpCas9，且相对于野生型SpCas9至少包含氨基酸取代H840A。示例性的野生型SpCas9包含SEQ ID NO:1所示氨基酸序列。在一些实施方案中，所述Cas9切口酶包含SEQ ID NO:2所示氨基酸序列。在一些实施方案中，所述融合蛋白中的Cas9切口酶能够在靶序列的PAM的-3位核苷酸(PAM序列5’端的第一个核苷酸为+1位)和-4位核苷酸之间形成切口。
在一些实施方案中，所述Cas9切口酶是能够识别改变的PAM序列的Cas9切口酶变体。本领域已知许多能够识别改变的PAM序列的Cas9切口酶变体。在一些实施方案中，所述Cas9切口酶是识别PAM序列5’-NG-3’的Cas9变体。在一些实施方案中，识别PAM序列5’-NG-3’的Cas9切口酶变体相对于野生型Cas9包含以下氨基酸取代H840A、R1335V、L1111R、D1135V、G1218R、E1219F、A1322R、T1337R，其中氨基酸编号参照SEQ ID NO:1。
本发明所述Cas9切口酶形成的切口能够导致靶序列形成具有3’末端的游离单链(3’游离单链)和具有5’末端的游离单链(5’游离单链)。
在一些实施方案中，本发明的融合蛋白中的所述逆转录酶可以衍生自不同来源。在一些实施方案中，所述逆转录酶是来源于病毒的逆转录酶。例如，在一些实施方案中，所述逆转录酶是M-MLV逆转录酶或其功能性变体。示例性的野生型M-MLV逆转录酶序列如SEQ ID NO:3所示。
在一些实施方案中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体
(a)包含在第155位、156位、200位和/或524位处的突变，例如包含选自F155Y、F155V、F156Y、D524N、N200C中任一种或其组合的突变，所述氨基酸位置参考SEQ ID NO:3；
(b)connection序列被缺失；和/或
(c)RNase H结构域被突变或缺失。
在一些优选实施方案中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体包含选自D524N突变，所述氨基酸位置参考SEQ ID NO:3。
在一些优选实施方案中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体的RNase H结构域被缺失。
在一些实施方案中，所述connection序列包含如SEQ ID NO:4所示的氨基酸序列。
在一些实施方案中，所述RNase H结构域包含如SEQ ID NO:5所示的氨基酸序列。
在一些实施方案中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体包含SEQ ID NO:9-15中任一项所述的序列。
在一些实施方案中，在所述融合蛋白中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体在N端或C端与核衣壳蛋白(NC)、水解酶(PR)或整合酶(IN)直接地或通过接头融合。所述核衣壳蛋白(NC)、水解酶(PR)或整合酶(IN)例如是来自M-MLV。
在一些实施方案中，所述核衣壳蛋白(NC)包含如SEQ ID NO:6所示的氨基酸序列。
在一些实施方案中，所述水解酶(PR)包含如SEQ ID NO:7所示的氨基酸序列。
在一些实施方案中，所述整合酶(IN)包含如SEQ ID NO:8所示的氨基酸序列。
在一些优选实施方案中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体在N端与核衣壳蛋白(NC)直接地或通过接头融合。
在一些优选实施方案中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体在C端与核衣壳蛋白(NC)直接地或通过接头融合。
如本文所用，“接头”可以是长1-50个(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或20-25个、25-50个)或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头可以是柔性接头，例如GGGGS、GS、GAP、(GGGGS)x 3、GGS和(GGS)x7等。例如，可以是SEQ ID NO:16所示接头。
在一些实施方案中，融合蛋白中的所述CRISPR切口酶位于所述逆转录酶的N端。在一些实施方案中，融合蛋白中的所述CRISPR切口酶位于所述逆转录酶的C端。
在本发明的一些实施方案中，本发明的融合蛋白还可以包含核定位序列(NLS)。一般而言，所述融合蛋白中的一个或多个NLS应具有足够的强度，以便在细胞的核中驱动所述融合蛋白以可实现其碱基编辑功能的量积聚。一般而言，核定位活性的强度由所述融合蛋白中NLS的数目、位置、所使用的一个或多个特定的NLS、或这些因素的组合决定。
在一些实施方案中，所述融合蛋白包含SEQ ID NO:19所示的氨基酸序列。
本发明的至少一种pegRNA中的引导序列(也称种子序列或spacer序列)被设置为与 靶序列具有充分的序列相同性(优选100％相同性)，从而能够通过碱基配对与靶序列的互补链结合，实现序列特异性靶向。
本领域已知多种适合用于基于CRISPR核酸酶(例如Cas9)的基因组编辑的gRNA的支架(scaffold)序列，这些可以用于本发明的pegRNA中。在一些具体实施方案中，所述gRNA的支架序列示于SEQ ID NO:17。
在一些实施方式中，所述引物结合序列被设置为与所述靶序列的至少一部分互补(优选与所述靶序列的至少一部分完全配对)，优选地，所述引物结合序列与靶序列所在DNA链中由切口导致的3’游离单链的至少一部分互补(优选与所3’游离单链的至少一部分完全配对)，特别是与所述3’游离单链的3’末端的核苷酸序列互补(优选完全配对)。当所述链的3’游离单链与所述引物结合序列通过碱基配对结合时，所述3’游离单链能够作为引物，以与所述引物结合序列紧邻的反转录(RT)模板序列作为模板，在融合蛋白中的逆转录酶的作用下进行反转录，延伸出对应于所述反转录(RT)模板序列的DNA序列。
所述引物结合序列取决于所使用的CRISPR切口酶在靶序列中形成的游离单链的长度，然而，其应当具有确保特异性结合的最少长度。在一些实施方案中，所述引物结合序列长度可以为4-20个核苷酸，例如长度为4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个核苷酸。
在一些实施方案中，所述引物结合序列被设置为具有不超过大约52℃的Tm(解链温度)。在一些实施方案中，所述引物结合序列的Tm(解链温度)为大约18℃-52℃，优选大约24℃-36℃，更优选大约28℃-32℃，更优选大约30℃。
计算核酸序列的Tm的方法为本领域公知，例如可以使用Oligo Analysis Tool在线分析工具计算。示例性的计算公式为Tm＝N G:C*4+N A:T*2，其中N G:C是序列中G和C碱基的数目，N A:T是序列中A和T碱基的数目。可以通过选择合适的PBS的长度来获得合适的Tm。或者，可以通过选择合适的靶序列来获得具有合适的Tm的PBS序列。
在一些实施方式中，所述RT模板序列可以是任意序列。通过上述反转录，其序列信息可以被整合进靶序列所在的DNA链(也即包含靶序列PAM的链)，再通过细胞的DNA修复作用，形成包含所述RT模板序列信息的DNA双链。在一些实施方案中，所述RT模板序列包含期望的修饰。例如，所述期望修饰包括一或多个核苷酸的取代、缺失和/或添加。例如，所述修饰包括一个或多个选自以下的取代：C至T取代、C至G取代、C至A取代、G至T取代、G至C取代、G至A取代、A至T取代、A至G取代、A至C取代、T至C取代、T至G取代、T至A取代；和/或包括一个或多个核苷酸的缺失，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸缺失；和/或包括一个或多个核苷酸的插入，例如1个至大约100个或更多个，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸插入。
在一些实施方式中，所述RT模板序列被设置为对应于靶序列切口下游的序列(例 如，与靶序列切口下游的序列的至少一部分互补)，并且包含期望的修饰。所述期望修饰包括一或多个核苷酸的取代、缺失和/或添加。例如，所述修饰包括一个或多个选自以下的取代：C至T取代、C至G取代、C至A取代、G至T取代、G至C取代、G至A取代、A至T取代、A至G取代、A至C取代、T至C取代、T至G取代、T至A取代；和/或包括一个或多个核苷酸的缺失，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸缺失；和/或包括一个或多个核苷酸的插入，例如1个至大约100个或更多个，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸插入。
在一些实施方式中，所述RT模板序列长度可以为大约1-300个或更多个核苷酸，例如长度为1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个、大约125个、大约150个、大约175个、大约200个、大约225个、大约250个、大约275个、大约300个核苷酸或更多个多核苷酸。优选地，所述RT模板序列长度为7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23个核苷酸。
在一些实施方案中，所述引导编辑系统还包括切口gRNA(nicking gRNA，用于产生额外切口)和/或含有编码所述切口gRNA的核苷酸序列的表达构建体，所述切口gRNA包含引导序列和支架(scaffold)序列。在一些优选实施方案中，所述切口gRNA不包含反转录(RT)模板序列和引物结合位点(PBS)序列。
本发明的切口gRNA中的引导序列(也称种子序列或spacer序列)被设置为与基因组中切口靶序列具有充分序列相同性(优选100％相同性)，从而能够将本发明所述融合蛋白靶向所述切口靶序列，并导致所述切口靶序列内的切口，所述切口靶序列与pegRNA靶向的靶序列(pegRNA靶序列)位于基因组DNA的相对链上。在一些实施方案中，所述切口RNA形成的切口和所述pegRNA形成的切口相距大约1个-大约300个或更多个核苷酸，例如相距1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个、大约125个、大约150个、大约175个、大约200个、大约225个、大约250个、大约275个、大约300个核苷酸或更多个核苷酸。在一些实施方案中，所述切口RNA形成的切口位于所述pegRNA形成的上游或下游(所述上游或下游均参照pegRNA靶序列所在的DNA链)。在一些实施方案中，所述切口gRNA中的引导序列与pegRNA靶序列在编辑事件发生后的相对链(经修饰)具有充分序列相同性(优选100％相同性)，从而所述切口gRNA仅靶向在pegRNA诱导的靶序列靶向及修改完成后才产生的切口靶序列。在一些实施方案中，所述切口靶序列的PAM位于所述pegRNA靶序列的互补序列内。
在一些实施方案中，所述的pegRNA和/或切口gRNA可以使用自加工系统对其序列进行精确加工。在一些具体实施方案中，所述pegRNA和/或切口gRNA的5’端连接 至第一核酶的3’端，所述第一核酶被设计为在所述pegRNA和/或切口gRNA的5’端切割所述融合物；和/或所述pegRNA和/或切口gRNA的3’端连接至第二核酶的5’端，所述第二核酶被设计为在所述pegRNA和/或切口gRNA的3’端切割所述融合物。所述第一或第二核酶的设计属于本领域技术人员的能力范围内。例如，可以参见Gao et al.,JIPB,Apr,2014；Vol 56,Issue 4,343-349。精确加工gRNA的方法例如可以参见WO 2018/149418。
在一些实施方案中，所述引导编辑系统包含至少一对pegRNA和/或含有编码所述至少一对pegRNA的核苷酸序列的表达构建体。在一些实施方案中，所述pegRNA对中的两种pegRNA被设置为靶向基因组DNA的相同链上的不同靶序列。在一些实施方案中，所述pegRNA对中的两种pegRNA被设置为靶向基因组DNA的不同链上的靶序列。在一些实施方案中，所述pegRNA对中的一种pegRNA的靶序列的PAM位于有义链，而另一种pegRNA的PAM位于反义链。在一些实施方案中，所述两种pegRNA的诱导的切口分别位于待修饰位点的两侧。在一些实施方案中，针对有义链的pegRNA诱导的切口位于待修饰位点的上游(5’方向)，针对反义链的pegRNA诱导的切口位于待修饰位点的下游(3’方向)。所述上游或下游相对于有义链而言。在一些实施方案中，所述两种pegRNA的诱导的切口相距大约1个-大约300个或更多个核苷酸，例如相距1-15个核苷酸。
在一些实施方案中，所述pegRNA对中的两种pegRNA被设置为导入相同的期望的修饰。举例而言，其中一种pegRNA被设置为在有义链导入A至G的取代，而另一种pegRNA则被设置为在反义链相应位置相应地导入T至C的取代。再举例而言，其中一种pegRNA被设置为在有义链导入两个核苷酸的缺失，另一种pegRNA则被设置为在反义链相应位置同样导入两个核苷酸的缺失。其它类型的修饰可以此类推。可以通过设计合适的RT模板序列来使分别靶向两条不同链的pegRNA实现相同的期望修饰。
在一些优选实施方案中，本发明所述pegRNA是epegRNA。epegRNA的构建可参考James W.Nelson et al.,Engineered pegRNAs improve prime editing efficiency.Nature Biotechnology volume 40,402–410(2022)，该文献通过引用并入本文。在一些实施方案中，所述epegRNA是带有3’-tevopreQ1-8nt linker修饰的epegRNA。
为了在不同生物体获得有效表达，在本发明的一些实施方式中，编码所述融合蛋白的核苷酸序列针对其基因组待进行修饰的生物体物种进行密码子优化。
密码子优化是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个或更多个密码子同时维持该天然氨基酸序列而修饰核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此， 可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在www.kazusa.orjp/codon/上可获得的密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。参见，Nakamura Y.等，“Codon usage tabulated from the international DNA sequence databases:status for the year2000.Nucl.Acids Res.，28:292(2000)。
三、修饰细胞基因组中靶序列的方法
另一方面，本发明提供了一种产生经遗传修饰的细胞的方法，包括将本发明的引导编辑系统导入至少一个所述细胞，由此导致所述至少一个细胞的基因组中靶序列的修饰。所述修饰包括一或多个核苷酸的取代、缺失和/或添加。例如，所述修饰包括一个或多个选自以下的取代：C至T取代、C至G取代、C至A取代、G至T取代、G至C取代、G至A取代、A至T取代、A至G取代、A至C取代、T至C取代、T至G取代、T至A取代；和/或包括一个或多个核苷酸的缺失，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸缺失；和/或包括一个或多个核苷酸的插入，例如1个至大约100个或更多个，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸插入
在另一方面，本发明还提一种产生经遗传修饰的细胞的方法，包括将本发明的引导编辑系统导入所述细胞。
在另一方面，本发明还提供经遗传修饰的生物体，其包含通过本发明的方法产生的经遗传修饰的细胞或其后代细胞。
在本发明中，待进行修饰的靶序列可以位于基因组的任何位置，例如位于功能基因如蛋白编码基因内，或者例如可以位于基因表达调控区如启动子区或增强子区，从而实现对所述基因功能修饰或对基因表达的修饰。可以通过T7EI、PCR/RE或测序方法检测所述细胞靶序列中的修饰。
在本发明的方法中，所述引导编辑系统可以通过本领域技术人员熟知的各种方法导入细胞。
可用于将本发明的引导编辑系统导入细胞的方法包括但不限于：磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射、病毒感染(如杆状病毒、痘苗病毒、腺病毒、腺相关病毒、慢病毒和其他病毒)、基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化。
可以通过本发明的方法进行基因编辑的细胞可以来自例如，哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物，包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。
在一些实施方式中，本发明的方法在体外进行。例如，所述细胞是分离的细胞，或 在分离的组织或器官中的细胞。
在另一些实施方式中，本发明的方法还可以在体内进行。例如，所述细胞是生物体内的细胞，可以通过例如病毒或土壤农杆菌介导的方法将本发明的系统体内导入所述细胞。
四、产生经遗传修饰的植物的方法
另一方面，本发明提供了一种产生经遗传修饰的植物的方法，包括将本发明的引导编辑系统导入至少一个所述植物，由此导致所述至少一个植物的基因组中的修饰。所述修饰包括一或多个核苷酸的取代、缺失和/或添加。例如，所述修饰包括一个或多个选自以下的取代：C至T取代、C至G取代、C至A取代、G至T取代、G至C取代、G至A取代、A至T取代、A至G取代、A至C取代、T至C取代、T至G取代、T至A取代；和/或包括一个或多个核苷酸的缺失，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸缺失；和/或包括一个或多个核苷酸的插入，例如1个至大约100个或更多个，例如1个至大约100个或更多个，例如1个、2个、3个、4个、5个、大约10个、大约20个、大约30个、大约40个、大约50个、大约75个、大约100个的核苷酸插入。
在一些实施方案中，所述方法还包括从所述至少一个植物筛选具有期望的修饰的植物。
在本发明的方法中，所述引导编辑系统可以本领域技术人员熟知的各种方法导入植物。可用于将本发明的引导编辑系统导入植物的方法包括但不限于：基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化、植物病毒介导的转化、花粉管通道法和子房注射法。优选地，通过瞬时转化将所述基因组编辑系统导入植物。
在本发明的方法中，只需在植物细胞中导入或产生所述引导编辑融合蛋白和gRNA即可实现对基因组的修饰，并且所述修饰可以稳定遗传，无需将编码所述编辑系统的组分的外源多核苷酸稳定转化植物。这样避免了稳定存在的(持续产生的)编辑系统的潜在脱靶作用，也避免外源核苷酸序列在植物基因组中的整合，从而具有更高生物安全性。
在一些优选实施方式中，所述导入在不存在选择压力下进行，从而避免外源核苷酸序列在植物基因组中的整合。
在一些实施方式中，所述导入包括将本发明的引导编辑系统转化至分离的植物细胞或组织，然后使所述经转化的植物细胞或组织再生为完整植物。优选地，在不存在选择压力下进行所述再生，也即是，在组织培养过程中不使用任何针对表达载体上携带的选择基因的选择剂。不使用选择剂可以提高植物的再生效率，获得不含外源核苷酸序列的经修饰的植物。
在另一些实施方式中，可以将本发明的引导编辑系统转化至完整植物上的特定部位，例如叶片、茎尖、花粉管、幼穗或下胚轴。这特别适合于难以进行组织培养再生的 植物的转化。
在本发明的一些实施方式中，直接将体外表达的蛋白质和/或体外转录的RNA分子(例如，所述表达构建体是体外转录的RNA分子)转化至所述植物。所述蛋白质和/或RNA分子能够在植物细胞中实现基因组编辑，随后被细胞降解，避免了外源核苷酸序列在植物基因组中的整合。
因此，在一些实施方式中，使用本发明的方法对植物进行遗传修饰和育种可以获得其基因组无外源多核苷酸整合的植物，即非转基因(transgene-free)的经修饰的植物。
一些实施方案中，所述方法还包括在升高的温度下培养已经导入所述引导编辑系统的植物细胞、组织或完整植物，所述升高的温度例如是37℃。
在本发明的一些实施方式中，其中所述被修饰的基因组区域与植物性状如农艺性状相关，由此所述修饰取代导致所述植物相对于野生型植物具有改变的(优选改善的)性状，例如农艺性状。
在一些实施方式中，所述方法还包括筛选具有期望的修饰和/或期望的性状如农艺性状的植物的步骤。
在本发明的一些实施方式中，所述方法还包括获得所述经遗传修饰的植物的后代。优选地，所述经遗传修饰的植物或其后代具有期望的修饰和/或期望的性状如农艺性状。
在另一方面，本发明还提供了经遗传修饰的植物或其后代或其部分，其中所述植物通过本发明上述的方法获得。在一些实施方式中，所述经遗传修饰的植物或其后代或其部分是非转基因的。优选地，所述经遗传修饰的植物或其后代具有期望的遗传修饰和/或期望的性状如农艺性状。
在另一方面，本发明还提供了一种植物育种方法，包括将通过本发明上述的方法获得的经遗传修饰的第一植物与不含有所述修饰的第二植物杂交，从而将所述修饰导入第二植物。优选地，所述经遗传修饰的第一植物具有期望的性状如农艺性状。
“农艺性状”特别是指作物植物的可测量的指标参数，包括但不限于：叶片绿色、籽粒产量、生长速率、总生物量或积累速率、成熟时的鲜重、成熟时的干重、果实产量、种子产量、植物总氮含量、果实氮含量、种子氮含量、植物营养组织氮含量、植物总游离氨基酸含量、果实游离氨基酸含量、种子游离氨基酸含量、植物营养组织游离氨基酸含量、植物总蛋白含量、果实蛋白含量、种子蛋白含量、植物营养组织蛋白质含量、除草剂的抗性抗旱性、氮的吸收、根的倒伏、收获指数、茎的倒伏、株高、穗高、穗长、抗病性、抗寒性、抗盐性和分蘖数等。
五、试剂盒
本发明还包括用于本发明的方法的试剂盒，该试剂盒至少包括本发明的引导编辑融合蛋白或所述引导编辑融合蛋白的表达构建体。该试剂盒还可以包含用于制备pegRNA的表达构建体及相关试剂。该试剂盒还可以包含用于将所述引导编辑系统导入生物体或生物体细胞的试剂。试剂盒一般包括表明试剂盒内容物的预期用途和/或使用方法的标 签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
材料与方法
1、载体构建
将编码PPE(SEQ ID NO:18)的基因片段通过PCR在M-MLV逆转录酶的相应位点进行点突变或删除相应片段，分别得到编码PPE-F155Y、PPE-F155V、PPE-F156Y、PPE-D524N、PPE-N200C、PPE-ΔRNase H、PPE-ΔRNase H-ΔConnection的基因片段，之后经过Gibson法连接，获得相应终载体。包含F155Y、F155V、F156Y、D524N、N200C的M-MLV逆转录酶序列分别如SEQ ID NO:9-13所示。M-MLV逆转录酶ΔRNase H的序列如SEQ ID NO:14所示。M-MLV逆转录酶ΔRNase H-ΔConnection的序列如SEQ ID NO:15所示。
此外，将编码核衣壳蛋白NC(SEQ ID NO:6)、水解酶PR(SEQ ID NO:7)或整合酶IN(SEQ ID NO:8)的基因片段经过植物密码子优化后，分别融合至编码PPE中M-MLV基因片段的5'端或3'端，之后经过Gibson法构建，获得相应终载体。同样，将编码核衣壳蛋白NC的基因片段经过植物密码子优化后融合至编码PPE-ΔRNase H的M-MLV逆转录酶ΔRNase H基因片段的5'端，获得PPE-ΔRNase H-NC(enhanced PPE，ePPE，氨基酸序列如SEQ ID NO:19所示)，之后经过Gibson法构建，获得相应终载体。具体载体如图1所示。
使用Gibson法将pegRNA片段(包括RT模板及PBS序列)构建至OsU3启动子启动的载体上，获得适用于水稻的OsU3-pegRNA构建体。
2、原生质体分离和转化
本发明中使用的原生质体来自于水稻中花11品种。
2.1水稻苗培养
水稻品种为中花11，种子先用75％乙醇漂洗1分钟，再用4％次氯酸钠处理30分钟，无菌水洗涤5次以上。放在M6培养基上培养3-4周，26℃，避光处理。
2.2原生质体分离
(1)剪下水稻茎秆，用刀片将其中间部分切成0.5-1mm的丝，放入0.6M的Mannitol溶液中避光处理10min，再用滤网过滤，将其放入50mL酶解液(0.45μm滤膜过滤)中，抽真空(压强约15Kpa)30min，取出后放置于摇床(10rpm)上室温酶解5h；
(2)加30-50mL W5稀释酶解产物，用75μm尼龙滤膜过滤酶解液于圆底离心管中(50mL)；
(3)23℃，250g(rcf)，升3降3，离心3min，弃上清；
(4)用20mL W5轻轻悬起细胞，重复步骤(3)
(5)加适量MMG悬浮，待转化。
2.3原生质体转化
(1)分别加所需转化载体各10μg于2mL离心管，混匀后，用去尖的枪头吸取200μL原生质体，轻弹混匀，加入220μL PEG4000溶液，轻弹混匀，室温避光诱导转化20-30min；
(2)加880μL W5轻轻颠倒混匀，250g(rcf)，升3降3，离心3min，弃上清；
(3)加1mL WI溶液，轻轻颠倒混匀，轻轻转至转移到流式管中，室温暗处培养40小时左右。
3、流式细胞分析
采用FACSAria III(BD Biosciences)仪器进行路试细胞分析，具体流程如下：
(1)检查鞘液桶和乙醇桶是否有充足的液体以及废液桶是否倾倒干净。开启稳压电源，仪器开机，打开荧光开关，打开BD FACSDiva Software软件，进入开机流程。
(2)断开气路和液路连接至鞘液桶上，确认闭合喷嘴在流动检测池相应位置，进行开机检测。
(3)更换合适孔径喷嘴，设置软件模式与实际喷嘴相符，开启激光，启动液流。
(4)选择新建程序，建立合适的实验方案。
(5)选择密度图，画两张散点图，一张为FSC/SSC散点图，一张为GFP/PE-Texas Red散点图。
(6)对FSC/SSC电压进行调整，使点分布于散点图中心位置附近。对FL1电压进行调整，使对照组原生质体群体分布于散点图中心位置附近，实验组原生质体群体(如转化GFP荧光蛋白的原生质体)分布于GFP荧光通道信号更强的位置。可以通过进一步调节补偿使两个群体之间的差异更显著。
(7)通过对照组确定门的界限，保证实验组原生质体群体位于界限范围之内且对照组原生质体群体位于界限范围之外。
(8)点击需分选的细胞群体，选择left sort，根据实验需要及目标细胞比例对分析条件及模式进行设置。
(9)依次上样读取度数，保存数据。
(10)关闭液流，更换闭合喷嘴，用FACS clean液体与无菌水清洗流动检测池，关闭软件及仪器，关闭稳压电源，释放仪器压力。
4、原生质体DNA提取与扩增子测序分析
4.1原生质体DNA提取
收集原生质体于2mL离心管中，利用CTAB法提取原生质体DNA(～30μL)，并利用NanoDrop超微量分光光度计测定其浓度(30-60ng/μL)，-20℃保存。
4.2扩增子测序分析
(1)利用基因组引物对原生质体DNA模板进行PCR扩增。20μL扩增体系包含4μL  5×Fastpfu buffer，1.6μL dNTPs(2.5mM)，0.4μL Forward primer(10μM)，0.4μL Reverse primer(10μM)，0.4μL FastPfu polymerase(2.5U/μL)，以及2μL DNA template(～60ng)。扩增条件：95℃预变性5min；95℃变性30s，50-64℃退火30s，72℃延伸30s，35个循环；72℃充分延伸5min，12℃保存；
(2)上述扩增产物稀释10倍，取1μL作为第二轮PCR扩增模板，扩增引物为含有Barcode的测序引物。50μL扩增体系包含10μL 5×Fastpfu buffer，4μL dNTPs(2.5mM)，1μL Forward primer(10μM)，1μL Reverse primer(10μM)，1μL FastPfu polymerase(2.5U/μL)，以及1μL DNA template。扩增条件如上，扩增循环数为35个循环。
(3)PCR产物于2％琼脂糖凝胶电泳分离，并利用AxyPrep DNA Gel Extraction kit对目的片段进行胶回收，回收产物利用NanoDrop超微量分光光度计进行定量分析；分别取100ng回收产物进行混合，并送生工生物工程有限公司进行扩增子测序文库构建及扩增子测序分析。
(4)待测序完成后，按测序引物对原始数据进行拆分，以WT作为对照，在3次重复试验的不同基因靶向位点上对产物的编辑类型及编辑效率进行比较和分析。
5.原生质体蛋白提取及蛋白免疫印迹(Western-Blot)
5.1原生质体蛋白质提取
(1)将培养48小时后的原生质体样品取出，颠倒混匀，12,000rpm离心8分
钟，弃掉上清液，加入50μL现配的蛋白提取液，涡旋振荡，使其混匀。
(2)将上一步骤中1.5mL的离心管在冰上静置30分钟，再次涡旋振荡混匀。
(3)低温离心机12,000rpm，4℃离心15分钟。
(4)用移液器取上清液，转移至一个新的1.5mL离心管中。
(5)从上述离心管中吸取2μL到新的1.5mL离心管中，加入18μL的双蒸水稀释。
(6)在上述体系中加入1mL Bradford 1×Dye Reagent，并充分振荡混匀后静置5分钟，用Eppendorf Biophotometer plus核酸蛋白测定仪对蛋白浓度进行初步测定。
(7)用蛋白提取缓冲液调整不同样品的浓度，加上样缓冲液10μL，振荡混匀，100℃金属浴5分钟使其变性。
(8)12,000rpm离心1分钟，用移液器吸取取上清液，使用SDS-PAGE胶电泳。
5.2蛋白免疫印迹(Western-Blot)
(1)配制8％的下层分离胶和5％的上层浓缩胶，上样，120V电泳1.5小时。
(2)提前剪好NC膜与滤纸，使用转膜缓冲液将其充分浸泡。
(3)本研究使用半干法进行转膜操作，将浸泡的滤纸，浸泡的NC膜，电泳完毕的SDS-PAGE胶，浸泡的滤纸从下到上放置。利用50mL圆底离心管来回滚动压实，尽量去除干净气泡。
(4)设置电流恒定为200mA，转膜1小时。
(5)将NC膜放入TBST缓冲液中，洗去转膜缓冲液，这一步骤约室温摇床上摇动5分钟。
(6)倒掉缓冲液，加入适量5％的脱脂牛奶(TBST配制)，在室温摇床慢速摇动孵育2小时，进行封闭。
(7)将封闭液倒掉，在新的5％脱脂牛奶中加入适当稀释比例的一抗，放于4℃摇床低转速过夜孵育。
(8)将一抗倒掉，使用TBST缓冲液洗涤NC膜3次，每次5分钟。
(9)加入含有稀释过的对应二抗的牛奶，室温孵育小时。
(10)将二抗倒掉，使用TBST缓冲液洗涤NC膜3次，每次5分钟，将NC膜封于特定的塑料袋中。
(11)将显色液A和显色液B(Tiangen)等量混匀，加入到上述塑料袋，孵育3分钟。
(12)排净塑料袋的显色液，放置于暗夹中，在暗室进行显影，显影时间根据蛋白丰度进行调整。
实施例1.使用报告系统筛选不同构建体
为了将上述多个改造快速且直观地筛选出效率有提升的构建体，在水稻原生质体中利用BFP-GFP报告系统进行初步筛选。通过流式细胞分析，结果如图2所示，表明PPE-ΔRNase H、PPE-NCv1和PPE-NCv2编辑效率相对原始PPE具有改善。因此选择这三个构建体进行后续内源靶点测试。
实施例2.不同PPE蛋白改造可提高引导编辑系统的编辑效率
为了测试上述筛选出的构建体对引导编辑系统的影响，对水稻16个内源靶点进行了测试。将这些构建体与pegRNA共转化至水稻原生质体细胞中，通过扩增子测序分析对引导编辑系统的编辑效率进行分析。结果如图3所示，表明在大多数测试位点中，PPE-NCv1，PPE-NCv2，PPE-ΔRNase H相对于PPE均有不同程度的效率的提升，其中PPE-NCv1提升幅度最高，PPE-NCv2和PPE-ΔRNase H次之。
实施例3.不同PPE蛋白改造的结合可进一步提高引导编辑系统的编辑效率
为了测试同时融合NC和删除RNase H(ePPE)是否可以进一步提升引导编辑系统效率。选取了12个位点进行测试，将这些构建体与pegRNA共转化至水稻原生质体细胞中，通过扩增子测序分析对引导编辑系统的编辑效率进行分析。结果如图4所示，表明在大部分位点中，ePPE(PPE-NCv1+ΔRNase H)编辑效率最高，效率从高到低依次为ePPE>PPE-NCv1>PPE-ΔRNase H>PPE。
实施例4.不同PPE蛋白改造后的蛋白表达量的测试
为了测试上述改造后PPE蛋白相比原始PPE蛋白是否在蛋白表达量上有所提高进而提升了其引导编辑效率，将相应蛋白转化水稻原生质体，提取原生质体蛋白后进行Western-Blot，结果如图5所示，表明PPE-NCv1、PPE-NCv2、PPE-ΔRNase H、ePPE蛋 白的表达量明显高于原始PPE蛋白表达量，且ePPE的蛋白表达量最高，与其引导编辑效率高低相对应。
实施例5.ePPE蛋白可有效提高大于15bp片段删除或插入的编辑效率
为了测试ePPE相比较于原始PPE蛋白除了对碱基替换或少数几个碱基删除或插入有效果外，还测试了该系统对稍大片段的插入或删除的效率。选取了18个位点进行测试，将这些构建体与pegRNA共转化至水稻原生质体细胞中，通过扩增子测序分析对引导编辑系统的编辑效率进行分析。结果如图6所示，表明所测试位点中，ePPE编辑效率最高，可有效实现18-34bp的插入及15-90bp的片段删除。
实施例6.ePPE蛋白可有效拓宽引导编辑系统范围
为了测试ePPE融合SpG核酸酶时相比较于原始PPE蛋白对其他PAM的靶向效果。选取了4个位点的NGC、NGA和NGG PAM进行测试，将这些构建体与pegRNA共转化至水稻原生质体细胞中，通过扩增子测序分析对引导编辑系统的编辑效率进行分析。结果如图7所示，所测试位点中ePPE编辑效率可有效拓宽引导编辑效果。
实施例7.ePPE在水稻愈伤中的编辑效率
为了进一步测试ePPE在稳定转化水稻愈伤中的突变效率。选取水稻的四个靶点进行测试。经过对抗性愈伤的鉴定，结果如图8所示，ePPE在愈伤中的编辑效率与其在原生质体中的测试效率一致，均比对照的PPE辑效率高，且编辑效率最高可达31.5％。
实施例8.ePPE创制抗除草剂水稻植物
通过靶向水稻OsALS基因的W548位点，将其突变为W548M，发现ePPE介导的突变效率为11.3％,PPE介导的效率只有0.6％。对杂合的突变体进行抗除草剂测试，发现其能够对烟嘧磺隆和甲咪唑烟酸，以及两者叠加都能产生很好的抗性，具体见图9。
实施例9.ePE在猪细胞中的编辑
为了测试ePPE在其他物种中的编辑效率，选取猪细胞的3个靶点进行测试(命名为ePE)。结果如图10所示，ePE相对于原始的PE处理的确有不同程度的效率提升。
实施例10.ePPE蛋白在引导编辑中的脱靶现象
为了确定ePPE是否显著增加了脱靶编辑的效率，分别检测了ePPE对pegRNA错配的耐受性以及与被测内源位点存在1-3个错配的潜在基因组脱靶位点的编辑效率。通过扩增子测序分析对引导编辑系统在内源靶点及潜在脱靶位点的编辑效率进行分析，结果如图11所示，相比于PPE，ePPE在大部分位点没有显著提高脱靶编辑的现象。
实施例11 ePPE与碱基编辑的对比
为了对比ePPE在引入碱基突变的编辑类型时与常用的碱基编辑器的编辑效率，选取7个水稻内源靶点将ePPE分别与A3A-PBE和PABE8e进行对比。扩增子测序分析结果如图12所示，受到PAM的限制，在不计入其他编辑位置的C或A时，引导编辑在精准编辑目标位点时具备更强的编辑优势，ePPE与碱基编辑实现功能的互补，在植物基因组编辑中实现更加灵活的应用。
实施例12 ePPE与pegRNA优化策略相结合进一步提高引导编辑效率
为了进一步提高植物引导编辑效率，将ePPE蛋白与dual-pegRNA策略、epegRNAs策略相结合。结果如图13所示，通过对不同epegRNAs的评估最终选择构建带有3’-tevopreQ1-8nt linker修饰的dual-pegRNA与PPE和ePPE共同转化原生质体，结合dual-pegRNA策略和epegRNAs策略的引导编辑效率存在进一步提高，ePPE-dual-epegRNA的组合相比于PPE-dual-pegRNA高出7.9倍。
序列表
>野生型SpCas9氨基酸序列(SEQ ID NO:1)
Figure PCTCN2022117258-appb-000001
>nCas9(H840A)氨基酸序列(SEQ ID NO:2)
Figure PCTCN2022117258-appb-000002
>野生型M-MLV-RT氨基酸序列(SEQ ID NO.3)
Figure PCTCN2022117258-appb-000003
>M-MLV-RT-connection(SEQ ID NO.4)
Figure PCTCN2022117258-appb-000004
>RT-RNase H (SEQ ID NO. 5)
Figure PCTCN2022117258-appb-000005
>NC (SEQ ID NO. 6)
Figure PCTCN2022117258-appb-000006
>PR (SEQ ID NO. 7)
Figure PCTCN2022117258-appb-000007
>IN (SEQ ID NO. 8)
Figure PCTCN2022117258-appb-000008
>M-MLV-RT-F155Y (SEQ ID NO. 9)
Figure PCTCN2022117258-appb-000009
>M-MLV-RT-F155V (SEQ ID NO. 10)
Figure PCTCN2022117258-appb-000010
Figure PCTCN2022117258-appb-000011
>M-MLV-RT-F156Y (SEQ ID NO. 11)
Figure PCTCN2022117258-appb-000012
>M-MLV-RT-D524N (SEQ ID NO. 12)
Figure PCTCN2022117258-appb-000013
>M-MLV-RT-N200C (SEQ ID NO. 13)
Figure PCTCN2022117258-appb-000014
Figure PCTCN2022117258-appb-000015
>M-MLV-RT-ΔRNase H(SEQ ID NO.14)
Figure PCTCN2022117258-appb-000016
>M-MLV-RT-ΔRNase H-ΔConnection(SEQ ID NO.15)
Figure PCTCN2022117258-appb-000017
>接头序列(SEQ ID NO:16)
Figure PCTCN2022117258-appb-000018
>gRNA支架(SEQ ID NO:17)
Figure PCTCN2022117258-appb-000019
>PPE(SEQ ID NO:18)
Figure PCTCN2022117258-appb-000020
Figure PCTCN2022117258-appb-000021
>ePPE (SEQ ID NO: 19)
Figure PCTCN2022117258-appb-000022
Figure PCTCN2022117258-appb-000023

Claims (15)

  1. 一种用于靶向性修饰植物基因组的引导编辑系统，其包含：
    i)引导编辑融合蛋白和/或含有编码所述引导编辑融合蛋白的核苷酸序列的表达构建体，其中所述引导编辑融合蛋白包含CRISPR切口酶和逆转录酶；和/或
    ii)至少一种pegRNA和/或含有编码所述至少一种pegRNA的核苷酸序列的表达构建体，
    其中所述至少一种pegRNA从5’至3’方向包含引导序列、支架序列、反转录(RT)模板序列和引物结合位点(PBS)序列，
    其中所述至少一种pegRNA能够与所述融合蛋白形成复合物并将所述融合蛋白靶向基因组中的靶序列，导致所述靶序列内的切口。
  2. 权利要求1的系统，其中所述CRISPR切口酶是Cas9切口酶，例如包含SEQ ID NO:2所示氨基酸序列。
  3. 权利要求1或2的系统，其中所述逆转录酶是M-MLV逆转录酶或其功能性变体。
  4. 权利要求1-3中任一项的系统，其中所述逆转录酶
    (a)包含选自F155Y、F155V、F156Y、D524N、D200C中任一种的突变，所述氨基酸位置参考SEQ ID NO:3；
    (b)connection序列被缺失；和/或
    (c)RNase H结构域被突变或缺失。
  5. 权利要求4的系统，其中所述逆转录酶包含SEQ ID NO:9-15中任一项所述的序列。
  6. 权利要求1-5中任一项的系统，其中在所述融合蛋白中，所述逆转录酶例如M-MLV逆转录酶或其功能性变体在N端或C端与核衣壳蛋白(NC)、水解酶(PR)或整合酶(IN)直接地或通过接头融合。
  7. 权利要求6的系统，所述核衣壳蛋白(NC)包含如SEQ ID NO:6所示的氨基酸序列，或所述水解酶(PR)包含如SEQ ID NO:7所示的氨基酸序列，或所述整合酶(IN)包含如SEQ ID NO:8所示的氨基酸序列。
  8. 权利要求6或7的系统，其中所述逆转录酶在N端与核衣壳蛋白(NC)直接地或通过接头融合，优选地，所述RNase H结构域被缺失的M-MLV逆转录酶的功能性变体在N端与核衣壳蛋白(NC)直接地或通过接头融合。
  9. 权利要求1-8中任一项的系统，其中pegRNA中的引导序列被设置为与靶序列具有充分序列相同性，从而能够通过碱基配对与靶序列的互补链结合，实现序列特异性靶向。
  10. 权利要求1-9中任一项的系统，其中所述pegRNA的支架序列包含SEQ ID NO:18所述序列。
  11. 权利要求1-10中任一项的系统，其中所述引物结合序列被设置为与所述靶序列 的至少一部分互补，优选地，所述引物结合序列与所述切口导致的3’游离单链的至少一部分互补，特别是与所述3’游离单链的3’末端的核苷酸序列互补。
  12. 权利要求1-11中任一项的系统，其中所述引物结合序列的Tm(解链温度)为大约18℃-52℃，优选大约24℃-36℃，更优选大约28℃-32℃，更优选大约30℃。
  13. 权利要求1-12中任一项的系统，其中，所述RT模板序列被设置为对应于切口下游的序列，并包含期望的修饰，所述修饰包括一或多个核苷酸的取代、缺失和/或添加。
  14. 权利要求1-13中任一项的系统，其中所述引导编辑融合蛋白包含SEQ ID NO:19所示氨基酸序列。
  15. 一种产生经遗传修饰的细胞的方法，包括将权利要求1-14中任一项的引导编辑系统导入至少一个所述细胞，由此导致所述至少一个细胞的基因组中靶序列的修饰。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118995664A (zh) * 2023-05-22 2024-11-22 江西农业大学 基于perv逆转录酶的先导编辑系统
CN120536504A (zh) * 2025-07-29 2025-08-26 江西农业大学 基于内源性多基因编辑的阿尔茨海默病模型猪的构建方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117720672B (zh) * 2024-02-07 2024-04-30 深锐(天津)生物医学有限公司 先导编辑系统及其应用
CN118440964A (zh) * 2024-05-17 2024-08-06 安徽农业大学 一种逆转录酶m-mlv rt基因序列及其应用
CN118652892A (zh) * 2024-08-21 2024-09-17 云南省第一人民医院 一种构建β地中海贫血CD17位点突变的HUDEP-2细胞株的引导编辑系统及方法
CN120173141B (zh) * 2025-05-21 2025-09-09 中国农业科学院植物保护研究所 一种用于引导编辑的蛋白其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018149418A1 (en) 2017-02-20 2018-08-23 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Genome editing system and method
WO2021080922A1 (en) * 2019-10-21 2021-04-29 The Trustees Of Columbia University In The City Of New York Methods of performing rna templated genome editing
WO2021082830A1 (zh) * 2019-11-01 2021-05-06 中国科学院遗传与发育生物学研究所 靶向性修饰植物基因组序列的方法
WO2021092204A1 (en) * 2019-11-05 2021-05-14 Spotlight Therapeutics Methods and compositions for nucleic acid-guided nuclease cell targeting screen
WO2021165508A1 (en) * 2020-02-21 2021-08-26 Biogemma Prime editing technology for plant genome engineering

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7657726B2 (ja) * 2019-03-19 2025-04-07 ザ ブロード インスティテュート，インコーポレーテッド 編集ヌクレオチド配列を編集するための方法および組成物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018149418A1 (en) 2017-02-20 2018-08-23 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Genome editing system and method
WO2021080922A1 (en) * 2019-10-21 2021-04-29 The Trustees Of Columbia University In The City Of New York Methods of performing rna templated genome editing
WO2021082830A1 (zh) * 2019-11-01 2021-05-06 中国科学院遗传与发育生物学研究所 靶向性修饰植物基因组序列的方法
WO2021092204A1 (en) * 2019-11-05 2021-05-14 Spotlight Therapeutics Methods and compositions for nucleic acid-guided nuclease cell targeting screen
WO2021165508A1 (en) * 2020-02-21 2021-08-26 Biogemma Prime editing technology for plant genome engineering

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
AREZI BAHRAM , HOGREFE HOLLY: "Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer.", NUCLEIC ACIDS RESEARCH, vol. 37, no. 2, 1 February 2009 (2009-02-01), GB , pages 473 - 481, XP002556110, ISSN: 1362-4962, DOI: 10.1093/nar/gkn952 *
CODON USAGE DATABASE, Retrieved from the Internet <URL:www.kazusa.orjp/codon>
GAO ET AL., JIPB, vol. 56, April 2014 (2014-04-01), pages 343 - 349
JAMES W. NELSON ET AL.: "Engineered pegRNAs improve prime editing efficiency", NATURE BIOTECHNOLOGY, vol. 40, 2022, pages 402 - 410, XP093043230, DOI: 10.1038/s41587-021-01039-7
MARTÍN-ALONSO SAMARA; FRUTOS-BELTRÁN ESTRELLA; MENÉNDEZ-ARIAS LUIS: "Reverse Transcriptase: From Transcriptomics to Genome Editing", TRENDS IN BIOTECHNOLOGY, vol. 39, no. 2, 8 July 2020 (2020-07-08), GB , pages 194 - 210, XP086446241, ISSN: 0167-7799, DOI: 10.1016/j.tibtech.2020.06.008 *
NAKAMURA, Y ET AL.: "Codon usage tabulated from the international DNA sequence databases: status for the year 2000", NUCL. ACIDS RES., vol. 28, 2000, pages 292, XP002941557, DOI: 10.1093/nar/28.1.292
SAMBROOK, J.FRITSCH, E.F.MANIATIS, T.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP4400594A4
YASUKAWA KIYOSHI, YANAGIHARA ITARU, FUJIWARA SHINSUKE: "Alteration of enzymes and their application to nucleic acid amplification (Review)", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 46, no. 5, 1 November 2020 (2020-11-01), GR , pages 1633 - 1643, XP093042055, ISSN: 1107-3756, DOI: 10.3892/ijmm.2020.4726 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118995664A (zh) * 2023-05-22 2024-11-22 江西农业大学 基于perv逆转录酶的先导编辑系统
WO2024240138A1 (zh) * 2023-05-22 2024-11-28 江西农业大学 基于perv逆转录酶的先导编辑系统
CN118995664B (zh) * 2023-05-22 2026-03-20 江西农业大学 基于perv逆转录酶的先导编辑系统
CN120536504A (zh) * 2025-07-29 2025-08-26 江西农业大学 基于内源性多基因编辑的阿尔茨海默病模型猪的构建方法

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