WO2018096356A1 - Procédés de désactivation génique conditionnelle - Google Patents

Procédés de désactivation génique conditionnelle Download PDF

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WO2018096356A1
WO2018096356A1 PCT/GB2017/053546 GB2017053546W WO2018096356A1 WO 2018096356 A1 WO2018096356 A1 WO 2018096356A1 GB 2017053546 W GB2017053546 W GB 2017053546W WO 2018096356 A1 WO2018096356 A1 WO 2018096356A1
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intron
sequence
gene
recombinase
nuclease
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Tilmann BUERCKSTUEMMER
Philippe Collin
Rodrigo SANTOS
Paloma GUZZARDO
Christina RASHKOVA
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Revvity Discovery Ltd
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Horizon Discovery Ltd
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Priority claimed from GBGB1717695.9A external-priority patent/GB201717695D0/en
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    • C—CHEMISTRY; METALLURGY
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

  • This invention relates to a system and method for generating conditional gene knock-out cells.
  • the system includes the use of an artificial intron to modify a target allele to permit conditional knock-out of the allele by disruption of the artificial intron.
  • Genome editing has been revolutionized by the discovery of the C ISP /Cas technology that enables the targeted introduction of double strand breaks into the human genome at unprecedented precision and efficiency 1"4 . Once the double-strand break has been
  • the cell will repair the break by one of two common pathways: in the presence of a homology template, the cell will use homology-directed repair or homologous
  • the cell will employ non- homologous end joining to repair the break 6 .
  • indels small insertions or deletions
  • Gene knockouts are strains containing a frameshift mutation or, ultimately, a stop codon in coding exons and thus represent complete loss-of-function mutants. They are fundamental for our understanding of gene function as they enable the study of the corresponding loss-of- function phenotype.
  • the homology donor was then used to establish genetically engineered cells by spontaneous homologous recombination (e.g. in mouse embryonic stem cells) or, more recently, by C ISP -assisted homologous recombination.
  • spontaneous homologous recombination e.g. in mouse embryonic stem cells
  • C ISP -assisted homologous recombination e.g. in C ISP -assisted homologous recombination.
  • degrons include the auxin/ mAID system 9 , the HaloPROTAC3/ HaloTag system 10 and the HCV protease inhibitor/ SMAShTag system 11 .
  • degrons has two major limitations: (i) The bi-allelic tagging of genes occurs at very low frequency and thus, it is difficult to obtain a cell line or an organism in which all copies of a gene have been conditionally inactivated and (ii) The insertion of foreign sequences within the coding region of a gene often disturbs protein folding or protein function. As a consequence, degron tagging may be less predictable than desired.
  • FLIP invertible intronic cassette
  • puroR puromycin resistance gene
  • polyadenylation signal pA
  • pA polyadenylation signal
  • This system has several shortcomings: (i) The cassette that is used as a donor is large (>2kb; the exact size not evident from the publication) and hence, the assembly of targeting constructs with homology arms is costly, cumbersome and time- consuming; (ii) The cassette contains an antibiotic resistance gene which is not always desired and (iii) Expression of the gene of interest is disrupted by a splice acceptor whose efficiency can vary depending on genomic context. Accordingly, there is a need for an alternative conditional gene knock out method.
  • the present invention is intended to address the disadvantages of the prior art.
  • the inventors have determined a method which is intended to significantly reduce the size of the artificial intron that is used and ensure that gene expression can be rapidly and efficiently abrogated in a wide range of mammalian genes.
  • the artificial intron size may be small so that homology donors can be easily assembled by gene synthesis; (ii) the approach does not require the use of antibiotic selection; (iii) the cassette disrupts gene expression completely, i.e. reveals a stop codon upon cassette activation; (iv) the cassette has a good "window of opportunity", so that protein levels are strongly decreased as a consequence of cassette activation; and (v) the approach is scalable and success is predictable for most human genes without too much prior insight into protein function or folding.
  • an artificial intron cassette that may contain as few as about 200 nucleotides. Insertion of this cassette into the coding exon of a gene does not disrupt gene function as the cassette is based on a natural intron sequence and the intron is thus removed by splicing. Addition of Cre recombinase (e.g. by addition of a recombinant adenovirus bearing Cre recombinase) leads to the deletion of critical elements within the intron that are required to maintain splicing, most importantly: the branch site. As a consequence, the cassette will no longer be removed and the ribosome will run into a stop codon, thus disrupting the expression of the gene of interest upon addition of Cre recombinase. The resulting cell line contains a frameshift and thus represents a complete gene knockout.
  • Described herein is a system that allows the generation of conditional gene knockouts on a large scale with the following features: (i) engineering is straightforward in diploid or even polyploid cells; (ii) the approach does not require any prior insight into the structure/ function of the targeted gene product; (iii) in the "off-state" of the cassette, the activity of the target gene product is largely unaffected; (iv) the cassette activation (transition to "on-state”) triggers a profound decrease in gene expression.
  • DECAI Degradation via Cre-regulated Artificial Intron
  • the present inventors have now developed a new conditional gene knock-out system that overcomes the above mentioned limitations.
  • the system uses an artificial intron sequence which, when introduced into the coding exon of a target gene, permits normal gene function because the artificial intron is based on a natural intron sequence and is thus removed by splicing.
  • the artificial intron includes one or more stop codons or a series of stop codons (one for each of the three possible reading frames) and an excisable or disruptable intron element.
  • this intron element is excised, intron function is disrupted and normal splicing of the intron is prevented.
  • the ribosome will then run into the stop codon, thus disrupting the expression of the target gene.
  • a method for generating an allele for conditional gene knock-out in a cell comprising a target gene comprising: introducing an artificial intron sequence into an exon of the target gene, the artificial intron sequence comprising:
  • the method may comprise introducing into the cell a sequence-specific nuclease that cleaves a sequence within the target gene thereby introducing the artificial intron sequence into the target gene.
  • the method may also include the step of introducing or activating a recombinase or nuclease in the cell thereby excising or disrupting the branch point and abrogating splicing of the artificial intron sequence.
  • the intron may be disrupted by applying two sequence-specific nucleases (e.g. Cas9 with two guide NAs targeting two sites flanking the branch point) to excise the branch point and thus abrogate splicing.
  • the guide RNA recognition sites flanking the branch point may also be identical, thus only requiring Cas9 along with a single guide RNA.
  • artificial introns are provided.
  • the artificial introns may be as described herein and may be used in any of the methods described herein.
  • the sequence of the artificial intron may comprise:
  • the sequence of the artificial intron may comprise two stop codons, more preferably three stop codons, which may be positioned in a series of three or more stop codons 5' to the branch point.
  • the artificial intron may contain a single branch point and may also lack related sequences that can take over branch point function once the single branch point has been removed.
  • the sequence-specific nuclease used to cleave the target gene may be an NA guided nuclease, or a DNA guided nuclease, a Zinc finger nuclease or a TALEN.
  • the RNA-guided nuclease may be Cas9 or an RNA-guided Argonaute.
  • the sequence-specific nuclease may be introduced as a protein, m RNA, or cDNA.
  • the first and second recombinase sites may be loxP sites or FRT sites.
  • the first (5') loxP site comprises at least one stop codon.
  • the first recombinase or nuclease site is positioned adjacent to the splice donor site.
  • the artificial intron sequence may be small, for example, about 200-
  • the artificial intron sequence comprises a nucleotide sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
  • the invention provides cells containing any of the artificial introns as described herein. Also provided are cells containing an artificial intron introduced according to any of the methods described herein.
  • the cells comprise an artificial intron in an exon of a target gene, the artificial intron having at least one stop codon positioned 5' of or within the first recombinase or nuclease site, and wherein the single branch site is flanked by a pair of recombinase or nuclease sites, the recombinase or nuclease sites being arranged such that the single branch site may be excised or disrupted, thereby abrogating splicing of the artificial intron.
  • a cell comprising an artificial intron in an exon of a target gene, the artificial intron having at least one stop codon positioned 5' to or within a first recombinase or nuclease site, and wherein a single branch site is flanked by a pair of recombinase or nuclease sites, the recombinase or nuclease sites being arranged such that the single branch site may be excised or disrupted, thereby abrogating splicing of the artificial intron, and wherein the artificial intron sequence also comprises a splice donor sequence and a splice acceptor sequence.
  • a cell comprising an artificial intron in an exon of a target gene, the artificial intron sequence comprising:
  • the cells used in the methods herein, or comprising the artificial intron sequences or nucleic acid constructs described herein may be eukaryotic cells, including mammalian cells such as haploid cells (e.g. the HAP1 cell-line), human cells, human induced pluripotent cells, human induced pluripotent stem cells, embryonic stem cells, or CHO cells or a T-cell (such as those for use in human therapy).
  • mammalian cells such as haploid cells (e.g. the HAP1 cell-line), human cells, human induced pluripotent cells, human induced pluripotent stem cells, embryonic stem cells, or CHO cells or a T-cell (such as those for use in human therapy).
  • the cells described herein may be used in therapy.
  • the cells described herein may be used to generate transgenic animals.
  • nucleic acid construct comprising an artificial intron sequence as described herein and which may be used in any of the methods described herein.
  • the nucleic acid construct further comprises 5' and 3' homology arms comprising sequences homologous to corresponding target gene sequences.
  • the nucleic acid constructs described herein may be part of a plasmid.
  • a vector comprising the artificial intron sequence or nucleic acid constructs as described herein.
  • Cre/loxP system has been used to create a variety of conditional KO mice. In most of these, a coding exon of the target gene was flanked with loxP sites. In the absence of Cre, gene expression is unaffected. Upon addition or activation of Cre, the intervening exon is deleted or inverted, resulting in a gene KO.
  • Cre could be applied by transient transfection or virus infection.
  • mice could already carry a Cre transgene, often coupled to a promoter that warrants expression in a specific tissue or at a certain stage of development.
  • Mice harbouring Cre are then crossed with strains harbouring alleles flanked with loxP sites. In that way, target gene expression can be disrupted in a tissue or developmental stage-specific manner, depending on which Cre mouse line was used for the intercross.
  • non-human transgenic animals whose genome comprises the artificial intron sequence described herein
  • mice In mice, this problem is often solved by creating a heterozygous conditional allele (e.g. by flanking an exon with loxP sites) and backcrossing heterozygous mice to homozygosity. This is unfortunately not possible in cell lines as they do not undergo sexual reproduction and hence, the conditional KO allele cannot be separated from the wild-type allele.
  • This problem is solved in an elegant way using the approach described herein: By targeting a coding exon with a g NA and introducing an artificial intron on one allele, the second allele is often disrupted as a consequence of Cas9 cleavage and repair by non-homologous end joining.
  • the resulting cell line will bear one allele harbouring the artificial intron and a second allele harbouring a frameshift mutation. For many essential genes, such lines will be viable. Hence, this solution alleviates the need for backcrossing and allows conditional KO model generation in one-go.
  • a gene Knockout(KO) is only desirable in a certain subtype or cells or at a certain developmental stage. In that case, it is feasible to engineer the total population of cells (e.g. all T cells) with the conditional KO cassette described herein.
  • Cre is then applied to a specific subset (e.g. the CD4-positive T cells) to inactivate the target gene selectively in this sub-population of cells.
  • Stem cell differentiation is often heterogenous or incomplete.
  • One way to improve the quality and outcome of the process would be inactivate certain genes at a specific point during the process and thereby steer cell differentiation in a certain direction.
  • a conditional Knockout (KO) allele into a pluripotency factor (e.g. Oct4/POU5Fl; as shown in Example 2) and thereby create an iPSC line in which pluripotency can be terminated at will.
  • this line will be subjected to differentiation by applying the appropriate cocktail of factors or by transducing with one or more master regulators as is known in the art.Once cells are starting to differentiate, one activates the conditional Oct4 allele (e.g. by addition of Cre recombinase).
  • Cre recombinase e.g. by addition of Cre recombinase
  • the cell may be an induced pluripotent stem cell and the target gene is Oct4.
  • conditional KO approach can be used to create more homogeneous differentiation protocols.
  • Genes that drive and control the maintenance of intermediate states of differentiation e.g. like pancreatic
  • the conditional KO cassette can be activated, leading to the death of progenitor cells that contaminate the culture. Reduction of tumorigenesis by suicide switches
  • conditional KO cassette described herein can be used to create a proliferation switch by targeting a gene involved in cell cycle (e.g. Cyclin-dependent Kinases). Blocking of proliferation can be carried out upon cell expansion to the required amounts for transplantation, or after stem cell differentiation into the desired cell type, and can be performed both prior and posttransplantation (different strategies can used for Cre delivery or Cre-activation).
  • a gene involved in cell cycle e.g. Cyclin-dependent Kinases
  • Certain fusion transcripts are commonly produced by cancer cells, and detection of these constitutes the diagnostic of certain cancer types.
  • Reference standards are materials that can be used as positive or negative controls during the diagnostic of patient material.
  • the conditional KO cassette described herein can be used to create transcript fusions to be used as reference material by allowing the hiding of a selection cassette (e.g. antibiotic resistance such as puromycin). This feature enables the generation and selection of fusion transcripts composed of an endogenous gene and a transgene, without any scar at the RNA level, as the synthetic intron is removed by slicing.
  • an element could represent an enhancer that is poorly described or annotated and altering this enhancer could have a profound impact on gene expression.
  • intron As the intron is actively removed by splicing, it is not present at the mRNA level and does therefore not impact transcription. Modifying the intron to contain an exogenous sequence (e.g. a molecular barcode) could represent an elegant way of "hiding" this sequence in the genome without leaving a trace in the transcriptome.
  • an exogenous sequence e.g. a molecular barcode
  • FIG. 1 NanoLuc expression from constructs containing synthetic intron configurations HAPl cells were transfected with a pcDNA3.1 plasmid expressing NanoLuc under the control of a CMV promoter. Cells were lysed 24h post transfection and analysed using the Nano-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. Median luminescence across 24 replicates is shown for each condition. As a control, expression levels from a plasmid containing the NanoLuc gene or the NanoLuc with the chimeric intron with no loxP sites were measured.. For sequence details of the different artificial introns see below SEQ I D NO:2 to SEQ ID NO: ll.
  • Figure 2 NanoLuc expression from constructs with chimeric intron at a different site
  • HAPl cells were transfected with a pcDNA3.1 plasmid expressing NanoLuc under the control of a CMV promoter. Cells were lysed 24h post transfection and analysed using the Nano-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. Median
  • Luminescence across 24 replicates is shown for each condition.
  • expression levels from a plasmid containing the NanoLuc gene or the NanoLuc with the artificial intron were measured.
  • FIG. 3 Genotyping CDK4-intron and METTL16-intron cell lines after Cre recombination Cells expressing CDK4-intron or M ETTL16-intron were infected with a recombinant retrovirus encoding Cre-ERT2. Upon enrichment of transduced cells, Cre was activated by adding ⁇ 4- hydroxitamoxifen (4-OHT) for three days. Genomic DNA from CDK4-intron and M ETTL16- intron cell lines treated with Cre recombinase and/or 4-OHT was used for a PCR amplifying the region around the insertion site of the artificial intron. Genomic DNA from wild type HAPl cells was used as a control. The expected sized of the PCR products are as follows. For CDK4,
  • HAPl WT 210bp
  • CDK4-intron before recombination: 411bp
  • CDK4-intron after recombination: 264bp.
  • M ETTL16 HAPl WT: 250bp
  • M ETTL16-intron before recombination: 451bp
  • M ETTL16-intron after recombination: 304bp.
  • Figure 4 Cell viability of CDK4-intron and METTL16 intron after Cre recombination
  • Figure 5 Approach for conditional gene inactivation using artificial introns.
  • an artificial intron is introduced into a coding exon of a gene.
  • the branch point in yellow
  • the branch point in yellow
  • the intron gets removed by splicing and leaves the m NA of the target gene intact.
  • Cre Upon recombination with Cre, the branch point is excised and the intron is inactivated.
  • FIG. 6 Identification of the artificial intron configuration that enables conditional gene inactivation.
  • HAPl cells were transfected with NanoLuc without intron ("no intron") or one harbouring the intron without further modification (“intron”).
  • intron an intron
  • cassette variants Var 1-5) were tested in which loxP sites positioning relative to the branch point varied. Note that we included two designs for each cassette, one in which the cassette was intact (“no Cre") and one in which the sequence between the loxP sites had been removed (“Cre”). NanoLuc levels were measured 24h post transfection using the Nano-Glo Dual assay. This data is also shown in Figure 1.
  • Variant 4 identified in Figure 6A was placed into various sequence contexts within the NanoLuc gene, reflecting one of four possible insertion sites: CAG-G, AAG-G, CAG-A and AAG-A. For each of these, two sites were evaluated. H EK293 cells were transiently transfected with these various constructs and NanoLuc luciferase activity was measured using the NanoGlo Dual assay 48h hours after transfection.
  • Figure 7 Cre-recombination leads to branch point excision.
  • A Genomic DNA was isolated from cells transfected with Cre (for CD46) or cells transduced with Cre-ERT2 and treated with 4-hydroxitamoxifen (4-OHT; for M ETTL16). Samples were genotyped using PCR primers specific for CD46 or M ETTL16. The right hand side of Figure 7A, regarding M ETTL16, is also shown in the right hand side of Figure 3.
  • B m NA was isolated from cells expressing Cre, reverse transcribed using oligo (dT) and analysed with primers specific for the CD46 cDNA.
  • FIG. 8 Artificial intron cassette activation leads to conditional gene inactivation.
  • a and B HAP1 cells bearing the artificial intron in CD46 were transfected with Cre recombinase. For Western blotting, cells were analysed using a CD46-specific antibody. For FACS, cells were stained with a CD46-specific antibody and analysed by flow cytometry.
  • C M ETTL16-DECAI cells were transduced with Cre-ERT2, treated with 4-OHT as indicated and stained with Crystal Violet. The data in Figure 8C is also shown in Figure 4, right hand side.
  • FIG. 9 Generation of Oct4-conditional knockout human iPS cells.
  • A Wild-type cells (+/+) or cells bearing the artificial intron in exon 1 of Oct4 (Oct4-DECAI #1 and Oct4-DECAI #2, created with two independent gRNAs) were transfected with Cre-ERT2 recombinase and 4-OHT or left untreated. Cells were fixed in 4% PFA and stained for Oct4 and SSEA4 using the specific antibodies. DAPI was used for nuclear staining. Images were acquired with an Olympus 1X83 microscope (10X magnification, ⁇ scale bars). For bright field images see Figure 14.
  • (B) Flow cytometry analysis of the cells before (No Cre) and after Cre treatment (+Cre) stained with Oct4 and SSEA4 specific antibodies.
  • FIG. 1 Homology donor templates for cell line generation. Homology donor templates used to engineer the CD46-DECAI, M ETTL16-DECAI and OCT4-DECAI cell lines are shown. The donors contain ⁇ 400bp right and left homology arms flanking the 201bp artificial intron cassette (underlined).
  • FIG. 11 Analysis of CD46 expression in CD46-DECAI and WT Hapl cells.
  • HAP1 wild-type and CD46-DECAI cells were transfected with a plasmid expressing Cre recombinase. Cells were then stained with a CD46-specific antibody and analysed by flow cytometry.
  • FIG. 12 Isolation of Cre-ERT2 expressing METTL16-DECAI clones. Crystal violet staining of single cell clones isolated from two M ETTL16-DECAI cell lines (Clone B5 and Clone G4). 12 clones of each cell line were isolated following transduction with Cre-ERT2 and these potentially Cre-ERT2 expressing clones were treated with 4-OHT to trigger cassette activation. Clones in which 4-OHT triggered cassette activation, and hence, cell death, are marked with a box.
  • Cre-recombination leads to a change in morphology of Oct4-DECAI cells.
  • a clear change in cell morphology is observed only in the Oct4-DECAI#l and Oct4-DECAI#2 cells.
  • Cre-recombination leads to branch point excision in Oct4-DECAI cells.
  • Genomic DNA was isolated from WT, Oct4-DECAI#l and Oct4-DECAI#2 cells that had been transfected with Cre-ERT2 and treated with 4-OHT, and from cells not transfected with Cre-ERT2. Samples were genotyped using PCR primers specific to either the cassette insertion site of Oct4-DECAI#l or Oct4-DECAI#2.
  • Introns are non-coding or intervening polynucleotide sequence of varying length, normally present within most eukaryotic genes and which are removed from a newly transcribed m RNA precursor by the process of splicing.
  • the process of splicing requires that the 5' and 3' ends of the intron be correctly cleaved and the resulting ends of the m RNA be accurately joined, such that a mature m RNA having the proper reading frame for protein synthesis is produced.
  • Many splice donor and splice acceptors sites meaning the sequences immediately surrounding the exon-intron- and intron-exon-boundaries, have been characterized and described and are known to the skilled artisan.
  • the sequence of mammalian introns begins with GT and ends with AG with a few minor introns starting with AT and ending with AC.
  • the artificial introns described herein comprise all the intron elements necessary for normal intron function. However, the artificial introns described herein have been modified by the inclusion of recombinase or nuclease sites so that intron function may be disrupted and gene function knocked-out.
  • the artificial intron described herein may be based on a natural intronic sequence with the intron elements that are necessary for normal intron function, including (i) a 5' splice donor, branch point and a 3' splice acceptor (ii) at least one of these intron elements, typically the branch point, is flanked by a pair of recombinase or nuclease sites; (iii) the 5'end of the cassette comprises at least one stop codon (preferably two stop codons, more preferable a series of three stop codons; one for each reading frame) which will be in frame with the target gene.
  • treatment with the recombinase or the nuclease will excise or invert the intron element, e.g. branch site, between the recombinase or nuclease sites, abrogate splicing of the intron causing the ribosome to run into a stop codon and terminate translation.
  • the intron element e.g. branch site
  • An artificial intron sequence derived from natural mammalian intron sequences may be used, for example based on a well-characterized chimeric intron found in the pCI-neo mammalian expression vector from Promega (Cat. No. E1841). This artificial intron is created by combining the 5' splice donor site from an intron of the human ⁇ -globin gene with the branch and
  • An artificial intron useful in this invention may be an intron which leads to comparable expression of the target gene of interest when placed in an exon of the target gene. Ideally, following disruption of the inserted intron, by recombinase addition or the like, gene expression should be significantly or even completely abrogated.
  • the artificial intron can be a) derived from a natural intron but may be modified by nucleotide substitutions, deletions and/or insertions, b) a chimeric intron composed of different intron sequences derived from one or more natural intron sequences of the gene of interest and/or of different genes, c) a de novo designed synthetic intron or d) any combination of the above.
  • Artificial intron sequences as described herein contain at least three conserved intron elements which are found in natural introns and are essential for splicing: a 5 ' splice site (also known as splice donor site), a branch point (or branch site) and a 3 ' splice site (also known as a splice acceptor) adjacent to a run of pyrimidines called a polypyrimidine tract. Recognition of these sites by the splicing machinery is followed by the excision of an intron. Splice donor site, splice acceptor site and branch point sequences are well known in the art and any may be utilized in the present invention.
  • splice donor and splice acceptor sites include the consensus splice donor and splice acceptor sites mentioned below and the sites shown in SEQ. ID NO. 1.
  • Efficient splice donor and acceptor sites suitable for this invention can be readily determined using techniques for measuring the efficiency of splicing. Intron splicing efficiency is readily determined by quantifying the spliced transcripts versus the full-length, unspliced transcripts that contain the intron(s), using methods known in the art.
  • the consensus sequences include sequences in the exon as well as the intron.
  • the splice sites as described herein include naturally occurring, engineered or synthetic, consensus or cryptic splice sites.
  • the branch point or branch site is usually located approximately between 10 and 60 nucleotides upstream of the splice acceptor and forms, during the splicing process via its conserved adenosine residue, a lariat structure with the splice donor.
  • one preferred branch point sequence is TACTAAC (Zhuang et al, PNAS 86, 2752-2756, 1989).
  • the branch point may be adjacent to or within the polypyrimidine tract of the splice acceptor site.
  • the artificial intron used in the constructs and methods of the invention has one or more stop codons in all possible 3 reading frames and/or has a nucleotide sequence length which is not dividable by 3 to prevent a complete read through of the intron sequence in case of a non-splicing event.
  • the artificial intron sequence may include stop codons present in the original intron sequence but may also include additional stop codon(s) positioned between the splice donor site and a first recombinase site.
  • the 5'end of the artificial intron preferably contains at least one stop codon, or two or three stop codons, that will be in frame with the target gene following successful recombination (and intron destruction).
  • treatment with the recombinase or the nuclease will destroy the intron and thus the ribosome will run into a stop codon and terminate translation.
  • Cre and Flp are the recombinases that are most commonly used in molecular biology. Cre can recognize LoxP sites (or variants thereof); Flp recognizes F T sites (or variants thereof). If a given site of interest is flanked by two parallel recombinase sites, then the addition of the cognate recombinase will trigger the excision of the intervening sequence with high efficiency and precision.
  • the key challenge was to add the recombinase sites (e.g. loxP sites as shown in the Exam ples herein) in a way that did not interfere with splicing.
  • the recombinase sites or nuclease sites should be positioned to flank an intron element, e.g. branch site, that is of critical importance to maintain splicing, such that when this intron element is excised or disrupted, by recombinase or nuclease addition, intron function is disrupted.
  • branch point sequence which was the prime candidate sequence for excision or disruption, is not very well defined and the sequence requirements are quite degenerate.
  • the first loxP site of the pair flanking the branch site which, typically, remains in the target gene following recombinase treatment, may be placed close to the 5' splice donor site, for example.
  • the first lox P site may comprise one or more stop codons. Variants of loxP sites are known to the person skilled in the art, for example, as shown here: https://en.wikipedia.org/wiki/Cre-
  • loxP site variants that have been described contain one or more stop codons.
  • the 5' loxP site may be placed in close proximity to the splice donor.
  • the 3' loxP site may also be placed in close proximity to the splice acceptor whilst retaining a functional polypyrimidine tract. Functionality can be assessed by common methods to measure splicing activity.
  • the branch site should be contained between the two loxP sites.
  • the present invention provides an artificial intron that may, advantageously, be small in size, for example about 200-250, 200-300, 200-400, 200-500, 200-600, 200-700 or 200-800 or 200- 1000 nucleotides in length. Insertion of this cassette into the coding exon of a gene does not significantly disrupt gene function as the cassette is based on natural intron sequences and is thus removed by splicing.
  • the consensus sequences include sequences in the exon as well as the intron.
  • the sequence necessary in the exon is MAG-G (where the hyphen denotes the intron).
  • Gene tagging can be conducted based on two concurring DNA repair mechanisms: Nonhomologous end joining or homology-direct repair.
  • homology-directed repair a donor nucleic acid construct is used that includes, in addition to the artificial intron sequence, homology arms of 100bp-5kb length. Importantly, both the 5' and the 3'end of the artificial intron needs to be flanked with homology arms. If such a nucleic acid construct is applied together with a nuclease, such as Cas9, then the artificial intron can be integrated into the exon of a target gene at reasonable efficiencies.
  • a nuclease such as Cas9
  • the resulting cell line will only represent a conditional knockout if all alleles in that cell are tagged. This is usually difficult as the efficiency of bi-allelic gene tagging is very low. This represents a limitation for the approach presented here, but even more so for competing approaches such as the degron tagging approach. Consequently, competing approaches have not been applied at scale.
  • the method presented here offers an attractive compromise to solve this problem, i.e. by targeting one allele with the conditional KO cassette and destroying the second allele with C ISP /Cas cleavage, followed by the introduction of a frameshift mutation through erroneous N H EJ. As a consequence, one would end up with one allele containing a frameshift mutation and the other one containing a conditional allele that could be inactivated upon recombinase addition.
  • hypomorphic alleles Such alleles would be usually referred to as “hypomorphic alleles" because they only retain gene function partially. Large scale mutagenesis in yeast has suggested that many essential genes tolerate such hypomorphic alleles.
  • Recombinases can be applied in many ways that are known to the skilled person. For instance, one could transiently transfect cells using lipofection or electroporation with an expression plasmid encoding the recombinase. Second, one can infect cells with a recombinant virus (retrovirus, lentivirus, adenovirus are most commonly used) encoding the recombinase. Third, one could create a cell line that stably and inducibly expresses the recombinase. Fourth, one could envisage applying the purified recombinase protein, either by itself (following electroporation) or as fusion with a cell-penetrating peptide (e.g. the arginine-rich motif of the H IV-1 TAT).
  • a recombinant virus retrovirus, lentivirus, adenovirus are most commonly used
  • guide RNA or "gRNA”
  • gRNA guide RNA
  • gRNA synthetic gRNA, gRNA obtained by in vitro transcription or gRNA expressed in cells from plasmid or PC product.
  • T is used in place of "U” in guide RNA sequences, as would be readily apparent to the skilled person.
  • the sequence of the intron cassette used in the examples herein is based on a well- characterized chimeric intron found in the pCI-neo mammalian expression vector from Promega (Cat. No. E1841). This chimeric intron is created by combining the 5' splice donor site from an intron of the human ⁇ -globin gene with the branch and 3 ' splice acceptor site of the intron of an immunoglobulin gene heavy chain variable region (SEQ I D NO:l).
  • the chimeric intron is shown with important splicing motifs highlighted. 5' and 3' splice sites are shown in bold, the branch site is double underlined and the polypyrimidine tract is underlined.
  • LoxP sites are 34 base pair sequences that are recognized by Cre recombinase to trigger a specific recombination event depending on their location and orientation. If the loxP sites are in parallel orientation, the sequence between the two sites is excised. To enable deletion of the branch site, two loxP sites were added to the cassette in parallel orientation flanking the branch site. Cre recombination will cause excision of the branch site and thus abrogate removal of the intron by the splicing machinery. As a consequence, the ribosome will read into the remaining cassette and encounter a premature stop codon that disrupts the gene of interest.
  • NanoLuc sequence is shown below.
  • the synthetic intron was inserted in the underlined site between the CAG and G.
  • the intron was inserted at the double underlines site between the AAG and G.
  • construct number 4 (SEQ ID NO:8 and SEQ ID NO:9) of the synthetic intron is the configuration that had no effect on NanoLuc expression with the cassette, but caused a profound ⁇ 30 fold reduction with the post-Cre configuration (compare NanoLuc + intron loxP v4 and NanoLuc + intron loxP post-Cre v4 conditions in Figure 1). Given these results, we decided to proceed with the version 4 cassette (SEQ ID NO:8) for all further experiments.
  • NanoLuc expression plasmids with the version 4 cassette inserted at the new site were electroporated in HAPl cells and NanoLuc expression was assayed after 24 hours ( Figure 2).
  • HAPl cells were transfected with Cas9, a g NA targeting the locus where the synthetic intron would be inserted and a homology donor (Table 1, SEQ ID NO: 13 and SEQ I D NO:14).
  • the site where the artificial intron would be inserted was selected following the exonic sequence requirements for splicing (SEQ I D NO:19 and SEQ I D NO:20).
  • the homology donor comprised the synthetic intron and 400bp homology arms upstream and downstream the insertion site (SEQ I D NO:21, SEQ I D NO:22).
  • Single cell clones containing the synthetic intron were isolated, and genotyped by PCR and Sanger sequencing (primers: Table 1, SEQ I D NO:15 to SEQ I D NO:18 and Sanger sequencing: SEQ ID NO:23, SEQ I D NO:24).
  • Genomic region surrounding the artificial intron insertion site is shown below. Exonic sequence is shown in lower case letters and intronic sequence in uppercase letter. The exonic splicing sequence motif is shown in bold. The guide RNA used during the cell line engineering is underlined.
  • CDK4 (SEQ I D NO: 19)
  • the sequence used as a donor template with ⁇ 400bp homology arms flanking the artificial intron is shown below.
  • LoxP sites are in lower case letters and the stop codons in all three reading frames are shown in italics.
  • the single nucleotide that was mutated to create a stop codon is in brackets.
  • CDK4 (SEQ ID NO:21)
  • the M ETLL16 and CDK4 clones containing the artificial intron were transduced with a retrovirus expressing Cre-ERT2 to induce recombination.
  • Cre-ERT2 encodes a Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) that requires tamoxifen or 4-Hydroxytamoxifen (4-OHT) for activity 14 .
  • ERT2 estrogen ligand-binding domain
  • 4-OHT 4-Hydroxytamoxifen
  • genomic DNA was isolated to verify presence or absence of the artificial intron by amplifying the insertion site by PCR ( Figure 3).
  • Cre recombination and addition of 4-OHT triggered the deletion of the sequence between the two loxP sites (the branch site) in both M ETTL16-intron and CDK4-intron cells.
  • Genomic DNA from CDK4-intron and M ETTL16-intron cell lines treated with Cre and/or 4-OHT was used for a PCR amplifying the region around the insertion site of the artificial intron.
  • the expected sized of the PCR products are as follows. For CDK4-intron (before recombination):
  • M ETLL16 is a highly essential gene in HAP1 cells and absence of this protein can be easily assessed by measuring cell viability.
  • equal numbers of cells for METTL16-intron and CDK4-intron were plated in six-well dishes following Cre-E T2 transduction and either treated or not treated with 4- OHT for a total of 6 days. Cells were then stained with crystal violet to assess total cell number as an approximation of cell viability (Figure 4).
  • the cassette we developed can be inserted into a reporter gene (NanoLuc) and insertion does not interfere with NanoLuc expression.
  • the cassette contains parallel loxP sites and removal of the sequences between the loxP sites triggers a ⁇ 30fold decrease in NanoLuc expression.
  • Cre recombinase e.g. by addition of a recombinant adenovirus bearing Cre recombinase
  • Cre recombinase leads to the deletion of critical elements within the intron that are required to maintain splicing, most importantly: the branch site.
  • the cassette will no longer be removed and the ribosome will run into a stop codon, thus disrupting the expression of the gene of interest upon addition of Cre recombinase.
  • the resulting cell line contains a frameshift and thus represents a complete gene knockout.
  • EXAM PLE 2 EXAM PLE 2:
  • gRNA sequences The gRNAs in Table 3 were used to insert the cassette. Table 3: gRNAs used to insert the cassette
  • NanoLuc assay HAP1 or HEK293 cells were transfected with a pcDNA3.1 plasmid encoding NanoLuc or NanoLuc containing artificial intron variants (see above) and pGL4.53[luc2/PGK] vector (Promega) for normalization. Cells were harvested 24h-48h post transfection and analysed using the Nano-Glo Dual Luciferase Assay System (Promega) according to manufacturer's instructions. Generation of cell lines
  • HAP1 cells were transfected with TurboFectin transfection reagent (OriGene).
  • TurboFectin transfection reagent OriGene
  • 0.8x10 s cells were seeded in a 6-well plate and transfected on the following day with l ⁇ g of Cas9 expression plasmid (#48137 from Addgene) , ⁇ g of U6 driven gRNA expression plasmid (Horizon Discovery), O ⁇ g of PCR product encoding the donor and O ⁇ g of plasmid encoding a blasticidin resistance gene.
  • 24h post transfection cells were treated with 20 ⁇ g/ml of blasticidin for 24h to eliminate untransfected cells. Single clones were obtained by limiting dilution. Positive clones were then identified by PCR screening.
  • iPS cells cultured in TeSR-E8 and Vitronectin XF were transfected with the P3 Primary Cell solution using the CM-138 pulse in the 4D-Nucleofector (Lonza).
  • 1.0x10 s cells were transfected with 2 ⁇ g of Cas9 expression plasmid, 2 ⁇ g of gRNA expression plasmid, 2 ⁇ g of plasmid encoding the donor and O ⁇ g of plasmid encoding a blasticidin resistance gene.
  • Cells were plated in culture media supplemented with ⁇ ROCKi (Y-27632, Abeam) for 24h, and then treated with 10 g/ml of blasticidin for 24h to eliminate untransfected cells. Recovered colonies were single cell plated after treatment with StemPro Accutase (Thermo Fisher Scientific) and clones were manually picked into 96wells for screening.
  • Genomic DNA was isolated using the QIAamp DNA Mini Kit (Qjagen) according to manufacturer's instructions. PCRs where done with the primers below using the GoTaq DNA Polymerase (Promega) following manufacturer's instructions. The primers in Table 4 were used for PCR.
  • TaqMan gene expression assays (Thermo Fisher Scientific) were used for each gene analysed. qRT-PCR experiments were performed using QuantStudio 6 Flex Real Time PCR System (Thermo Fisher Scientific). All values were normalized to an internal control (GAPDH) and brought to power of 2. Each target's value was then compared to the highest value of that target across all samples. All samples were done in technical triplicates and the median of these is displayed. The following TaqMan gene expression assays were used: Gapdh
  • Hs03929097_gl Oct4 (Hs00999632_gl), Nanog (Hs04399610_gl), Sox2 (Hs01053049_sl), Cdx2 (Hs01078080_ml), Eomes (Hs00172872_ml) (all from Thermo Fisher Scientific).
  • Approximately lxlO 7 HEK392T cells were transfected with 1.7 ⁇ g pAdvantage plasmid (Promega), 2.6 ⁇ g VSV-G expression vector, 4 ⁇ g gag-pol expression vector and 6.6 ⁇ g CreERT2 expression vector, using TurboFectin transfection reagent (OriGene). Medium was changed 24 hours after transfection. Viral supernatant was collected 48 and 72 hours after transfection, and used to transduce the M ETTL16-DECAI cells. Since the Cre-ERT2 retrovirus also encoded a PGK-PuroR cassette, cells containing Cre could be enriched for by selecting with 0.3 ⁇ g/ml puromycin. FACS, Western Blotting and Immunocytochemistry
  • HAP1 experiments cells were trypsinized and washed with PBS. Cells were stained with a CD46-specific antibody (APC-CD46; catalogue #564253; BD Biosciences) in FACS buffer (5% FCS in PBS) for 30 minutes and excess antibody was removed by washing with PBS. Cells were analysed by flow cytometry using the BDLSR Fortessa. For the human iPS cell experiments, cells were fixed and permeabilized using the
  • Cytofix/Cytoperm kit (BD) according to manufacturer's instructions. Cells were then stained using the following directly conjugated antibodies: Alexa Fluor ® 488 anti-Oct4 (653706, BioLegend) and PE/Cy7 anti-human SSEA-4 (330420, BioLegend). Cells were analysed using the iQue Screener PLUS (IntelliCyt). Data was analysed using the FlowJo software.
  • lysates were prepared using Frackelton buffer (lOm M Tris/HCI pH7.5, 50mM NaCI, 30mM sodium pyrophosphate, 1% Triton X-100, 50mM NaF and protease inhibitors). Lysates were separated on 10% SDS-PAGE and blotted on Nitrocellulose membranes. Membranes were stained with a primary antibody (anti-CD46, abl08307, Abeam) and a secondary antibody (anti-rabbit, 111-035-003, Jackson Immunoresearch Europe) and visualized using ECL reagent (Thermo Fisher Scientific).
  • the PSC 4-Marker Immunocytochemistry Kit (Thermo Fisher Scientific, A24881) was used. Briefly, iPS cells were fixed using 4% PFA for 15min,
  • variant 1 and 2 were well tolerated, yet we did not observe any reduction in gene expression upon Cre recombination, suggesting that alternative branch points had taken over.
  • variant 4 Figure 5C was most fit for purpose in as much as it was well tolerated in the "off-state” and triggered a profound decrease ( ⁇ 14 fold) in NanoLuc expression upon cassette activation.
  • Introns of the GT-AG category such as the one we chose, have a preference for CAG or AAG upstream of the splice donor and G or A
  • the frequency at which the cassette was incorporated was higher in the essential gene (M ETTL16) than in the non-essential gene (CD46). While this may well be a coincidence, it is plausible to assume higher targeting frequencies for essential genes as other editing events (e.g. the accumulation of indels) may be subject to negative selection.
  • Human iPSCs are pluripotent and their pluripotency depends on the expression of the transcription factor Oct4/POU5Fl 18 .
  • Oct4 Inactivation of Oct4 triggers the collapse of the core transcriptional network leading to rapid loss of pluripotency. This is associated with drastic changes in colony morphology and loss of other pluripotency markers, such as Nanog, Sox2 and SSEA4.
  • We created homology donors for Oct4/POU5Fl 400bp homology arms flanking the DECAI cassette) and targeted two independent sites in exon 1 (Figure 10). Clonal cell lines were established and genotyped by PCR. Both homozygous and heterozygous cell lines were achieved (Table 5).
  • Oct4- DECAI cells lost Oct4 expression, and consequently, SSEA4 (Figure 9A-B), Nanog and Sox2 expression (Figure 9C). They also changed their morphology quite dramatically ( Figure 13), indicating that these cells had lost their pluripotency.
  • the induced knockout of Oct4 resulted in differentiation of human iPSCs with a significant increase in transcription of Cdx2 and Eomes ( Figure 9C), genes associated with trophoblast and endoderm lineages 1 ,20 .
  • Cre/loxP recombination at the genomic DNA level ( Figure 14). Altogether, this highlights the feasibility of our approach for conditional gene inactivation and suggests its applicability in more physiological settings. Discussion
  • the artificial intron approach presented here also has some conceivable shortcomings.
  • insertion of the artificial intron may dysregulate endogenous splicing events, leading to constitutive gene inactivation.
  • CDK4 gene which is nonessential in HAP1 cells
  • CDK4 expression data not shown.
  • a more detailed analysis of these cells revealed that the intron was successfully removed by splicing, but some erroneous splicing events led to the assembly of a non-functional mRNA that was most likely degraded by non-sense mediated decay.
  • Cre-ERT2 by retroviral transduction for M ETTL16-DECAI and OCT4-DECAI activation. While this is possible, we noticed some shortcomings, mainly: (i) Cre-ERT2 expression can be leaky and already occur in the absence of 4-OHT (data not shown) and (ii) Cre-ERT2 expression can be silenced, especially when Cre-ERT2 is delivered via retroviral infection. A more elegant approach would be to express Cre-ERT2 from a safe-harbour locus such as AAVS1 or ROSA26.
  • CRISPRi CRISPR interference
  • a catalytically dead Cas9 directed by a guide RNA will suppress expression of the target gene by sterically hindering transcription initiation/ elongation or by the action of a silencing effector domain fused to Cas9 22,23 . While this strategy has the advantage of being reversible, it is difficult to reach complete inhibition of the target gene expression.
  • inducible CRISPR systems There are several iterations of these systems ranging from split Cas9 molecules that form a catalytically active molecule upon a certain stimulus 24 or control of Cas9 or gRNA expression by inducible promoters 25 . The efficiency of these methods will depend on the expression level of the gRNA and will likely show a variety of effects across cells in the sample since the editing induced by Cas9 will not happen in a predictable manner. This contrasts with the DECAI method described herein which will lead to a predictable modification and disruption of expression upon Cre recombination.
  • Degrons are drug-regulatable domains that allow one to modulate the abundance of the degron tagged protein by adding or withdrawing a small molecule.
  • Popular degrons include the auxin/ mAI D system 9 ,the HaloPROTAC3/ HaloTag system 10 and the HCV protease inhibitor/ SMAShTag system 11 .
  • At least some of the degrons that are commonly used are much faster in degrading the client protein (with a half-life of minutes 9 rather than hours or days) as they lead to the recruitment of E3 ligases that actively degrade the protein population that is present.
  • the degron tags have the advantage of allowing reversible and tuneable degradation of the target proteins.
  • the use of degrons has two major limitations: (i) the bi-allelic tagging of genes occurs at very low frequency and thus, it is difficult to obtain a cell line or an organism in which all copies of a gene have been conditionally inactivated and (ii) the insertion of foreign sequences within the coding region of a gene could disturb protein folding or protein function.
  • degron tagging can be cumbersome and may not be compatible with high-throughput applications.
  • the approach described herein is more suitable for serial production of conditional knockouts, whereas degron tagging may be the method of choice if the protein is well understood and inefficient clone recovery is less of a constraint.
  • the DECAI method described herein offers a convenient and efficient alternative for generating conditional knockout alleles in mammalian cells.
  • HaloPROTACS Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins. ACS Chem Biol 10, 1831-1837, doi:10.1021/acschembio.5b00442 (2015).
  • Burckstummer, T. et al. A reversible gene trap collection empowers haploid genetics in human cells. Nat. Methods 10, 965-971 (2013).
  • Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-51 (2013).

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Abstract

La présente invention concerne un système et un procédé de génération de cellules de désactivation génique conditionnelle. Le système comprend l'utilisation d'un intron artificiel pour modifier un allèle cible afin de permettre une désactivation conditionnelle de l'allèle par destruction de l'intron artificiel. En particulier, l'invention concerne un procédé de génération d'un allèle pour une désactivation génique conditionnelle dans une cellule comprenant un gène cible, le procédé consistant à : introduire une séquence d'intron artificiel dans un exon du gène cible. L'invention concerne également des cellules contenant un intron artificiel introduit selon le procédé. L'approche a une large utilité pour l'activation génique conditionnelle et pourrait être utilisée pour étudier les phénotypes de perte de fonction de gènes essentiels.
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