Patent Application
20240229080
The present invention relates to the field of controlled gene activation and inactivation.
Despite the revolutionary advancement with CRISPR technology, the generation of conditional allele has not been as easy as knockout and knock-in alleles. To study essential genes such as housekeeping or developmentally required genes, conditional knockout (cKO) approach, allowing spatiotemporal control of gene knockout, is ideal as, otherwise, a simple knockout approach may cause early developmental lethality. For many years, recombinase systems, like the Cre/LoxP system, have been widely utilized to construct cKO alleles, by inserting two (LoxP) recombination sites in the adjacent introns of essential exon (s). The making of conditional alleles (so called ‘floxed’ alleles) in mice traditionally involved the use of mouse embryonic stem cells (mESCs) (Bouabe & Okkenhaug. Methods in Molecular Biology 315-336 (2013)). Recently, cKO alleles have also been generated via CRISPR/Cas9-mediated insertion of LoxP sites, which turned out to be rather challenging even with additional refinements (Gurumurthy et al. Genome Biology (2019) 20:171).
Conditional intron approaches have been attempted in the past as it allows a simple insertional mutagenesis with a fixed universal conditional intronic cassette (Economides et al. Proceedings of the National Academy of Sciences 110, E3179-E3188 (2013); Andersson-Rolf et al. Nature Methods 14, 287-289 (2017); WO 2017/203275 A1; Guzzardo et al. Scientific Reports 7, (2017); WO 2018/096356 A1), but it has not been well utilized in animal models due to their long length (Economides et al. and Andersson-Rolf et al.) or an unexpected hypomorphic effect, a lowered gene expression of the targeted gene even before triggering the insert (Guzzardo et al.).
It is a goal of the invention to provide a conditional allele with reduced or no hypomorphic effect and that is easy to utilize in various organisms.
The present invention provides a genetic element comprising: a splice donor site, a first recombinase recognition site, a splice branch point, a second recombinase recognition site, a splice acceptor site, wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site.
The invention further provides a genetic vector comprising the genetic element.
Further provided is a method of providing a cell with a conditionally de-activatable gene, comprising providing a cell, introducing the genetic element into an exon of a gene in a cell, or introducing a gene with the genetic element into a cell.
Further provided is a cell comprising a gene with two or more exons and at least one intron, wherein the intron comprises the genetic element.
Also provided is a non-human animal comprising one or more cells of the invention.
Further provided is a method of inactivating expression of a functional gene in a cell or non-human animal, comprising providing a cell or a non-human animal of the invention and activating recombination at the recombinase recognition sites in the cell or in a cell in the non-human animal.
Further provided is a method of investigating the function of a gene, comprising inactivating a functional gene according to the method of the invention and comparing the inactivated gene's effect in the cell or non-human animal to a cell or nonhuman animal without inactivation of the gene or to a cell or non-human animal without the genetic element.
Also provided is a kit suitable for integrating an intron into a target gene comprising a genetic element of the invention and a Cas encoding nucleic acid.
All the above aspects of the invention are related and the following detailed description of particular embodiments relate to all aspects alike, even when descripted with focus on one embodiment. E.g. a description of the genetic element also relates to the method of incorporating it into a cell or animal or using it and the cells, animals or kits comprising the element. Descriptions of methods relate to the genetic element in terms of suitabilities of the genetic element for such methods. Descriptions of methods also relate to products of the methods as obtainable by the methods. The kit may comprise any part or component as used in the methods and may be suitable to perform the methods. The kit may of course also be used in the methods.
The present invention provides a conditional intron system that is suitable for conditional knock-out approaches. The intron can be incorporated into an allele and activated at a time of choice in the living system during any time of biological development.
The generation of conditional allele using CRISPR technology is still challenging. The invention provides a Short Conditional intrON (also termed “SCON”) that enables a rapid generation of conditional allele. For example, it can be used with a simple one-step zygote injection, among other uses. SCON has conditional intronic function in various organisms, including vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.
In particular, the invention provides a genetic element comprising: a splice donor site, a first recombinase recognition site, a splice branch point, a second recombinase recognition site, a splice acceptor site, wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site. The term “distance” as used herein refers to a number of nucleotides in length between the sites, not counting the nucleotides of the sites themselves. E.g. the nucleotides after “CTGAC”, as an example of a branch point, and up to but excluding “AG”, as an example of a splice acceptor, site can be counted. In preferred embodiments, the distance between the splice branch point and the splice acceptor site is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 52, 53, 54 or 55 nucleotides. A preferred distance range is 35 to 50 nucleotides, e.g. 40 to 48 nucleotides, in length between the splice branch point and the splice acceptor site.
The genetic element of the invention can also be referred to as “nucleic acid construct” or “artificial intron” or “SCON”.
The genetic element can be a nucleic acid molecule, preferably DNA. It may also be RNA. RNA genetic elements are preferably reverse transcribed into DNA before or during use. In case of RNA genetic elements, all disclosures of DNA sequences herein in preferred elements read on the corresponding RNA sequence, in particular with T being U. The nucleotides A, G, C T/U refer to the function in the genetic code. Chemically, the nucleotides can be A, G, C T/U nucleotide molecules or nucleotide molecule moieties (in a polynucleotide molecule), respectively, or any other nucleotides corresponding to the same code, such as modified nucleotides, like pseudouridine.
Any sequence herein may also be provided in the complementary reverse sequence, i.e. the anti-sense strand to the sequence provided herein. The genetic element as for use can be provided by generating the sense strand to such an anti-sense strand.
The genetic element may be provided as single strand or double strand nucleic acid, double strands are preferred for increased stability.
It is also beneficial when the splice branch point is close to one of the recombinase recognitions sites, in particular the second recombinase recognition site. In preferred embodiments, the splice branch point is at a distance of 0 to 11 nucleotides in length, e.g. at a distance of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length, from the second recombinase recognition site. The splice branch point may be directly adjacent to the second recombinase recognition site. In cases where there are more than one splice branch points, e.g. two or more splice branch points, the splice branch point closer to the second recombinase recognition site is considered.
In further preferred embodiments the splice donor site is at a distance of 80 to 5000 nucleotides in length, preferably 100 to 4500 nucleotides in length, from the splice acceptor site. Particular preferred nucleotides in length (nt) between the splice donor site and the splice acceptor site are 110 to 4000 nt, preferably 120 to 3500 nt, 130 to 3000 nt, 140 to 2500 nt, 150 to 2000 nt, 160 to 1500 nt, nucleotides in length. Further ranges are up to 1000 nt, up to 800 nt, up to 500 nt, up to 400 nt or up to 300 nt or up to 190 nt. Upper range limits can be combined with any of the lower range limits, such as 110 to 400 nt, preferably 120 to 350 nt, 130 to 300 nt, 140 to 250 nt, 150 to 200 nt, 160 to 190 nt, nucleotides in length. A shorter distance, such as below 500 nt or below 400 nt have the advantage of reduced costs and time efforts when handling the genetic element.
Preferably, a sequence of 10 nucleotides directly 5′ adjacent to the splice acceptor site contains at least 8, preferably at least 9 or 10, pyrimidine nucleotides, preferably of which at least 3 nucleotides are C and/or preferably at least 3 nucleotides are T. This sequence may also be referred to as a “polypyrimidine tract”. Substantial purine base (A, G, but especially A) content in this sequence may lead to an unwanted hypomorphic effect. Preferably the at least 8 pyrimidine nucleotides are directly 5′ adjacent to the splice acceptor site.
In particular preferred embodiments of all aspects of the invention, the splice donor site comprises the nucleic acid sequence GTPuAG, with Pu being a purine base e.g. A or G, such as in GTAAG and GTGAG. A T nucleotide may follow the sequence GTPuAG in preferred embodiments; accordingly, the genetic element may comprise the sequence GTPuAGT, such as GTAAGT or GTGAGT at a splice donor site. The GT nucleotides in the sequence GTPuAG are most significant. Preferably the splice donor site contains the nucleotides GT. In preferred embodiments of the invention all distances given above for distances between the splice donor site and a further site may also be given as distance from the GT nucleotides of the splice donor site to that further site, plus 3 nucleotides (accounting for the PuAG sequence). The splice donor site may be preceded by the sequence MAG with M being C or A. Further splice donor sites are reported in Ohshima et al., J. Mol. Biol. 195, 247-259, 1987.
In preferred embodiments, the splice branch point comprises the nucleic acid sequence CTPuAPy, wherein Pu refers to a purine base, such as G or A and Py refers to a pyrimidine base, such as C and T or U. The splice branch point may comprise the sequences CTGAT, CTGAC, CTAAT or CTAAC. The A between Pu and Py is most significant. Preferably the splice branch point comprises an A, preferably between a Pu and a Py. In preferred embodiments of the invention all distances given above for distances between the splice branch point and a further site may also be given as distance from the A nucleotide of the splice branch point to that further site, plus 1 nucleotide for following further sites in 3′ direction (accounting for the Py nucleotide); or plus 3 nucleotides for preceding further sites in 5′ direction (accounting for the CTPu sequence). For example, the distance between the A of the splice branch point and the splice acceptor site can be 11 to 57 nucleotides in length, such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 52, 53, 54, 55 or 56 nucleotides. A preferred distance range is 36 to 51 nucleotides, e.g. 41 to 49 nucleotides, in length between the A of the splice branch point and the splice acceptor site. Further splice branch points are disclosed in Zhuang et al., PNAS 86, 2752-2756, 1989.
In preferred embodiments, the splice acceptor site comprises the sequence AG. In some embodiments, there may be a pyrimidine nucleotide (“Py”), in particular C or T (or U in case of RNA), directly preceding the splice acceptor site, preferably AG, such as in the sequence PyAG, in particular CAG or TAG. The AG nucleotides in the splice acceptor site are very efficient for splicing and are preferably contained in the splice acceptor site. Preferably the splice acceptor site comprises an A nucleotide. In preferred embodiments of the invention all distances given above for distances between the splice acceptor site and a further site may also be given as distance from the A nucleotide of the splice acceptor site to that further site. Further splice acceptor sites are disclosed in Seif et al., Nucleic Acids Res. 6 (10), 3387-98, 1979.
The expression “recombinase recognition site” is also referred to as just “recombinase site” or recombinase target site or recombinase recognition targets. Preferably the first and second recombinase recognition sites are selected from a tyrosine recombinase site (recombinase sites of tyrosine-type site-specific recombinases (T-SSRs)). Example recombinases and sites are Flp (flippase), which binds to flippase recognition target (frt) sites. Preferred sites are lox (Cre) and frt (Flp). A preferred lox site is loxP that is derived from a bacteriophage P1 sequence. Other lox sites are e.g. Lox 511, Lox 5171, Lox 2272, M2, M3, M7, M11, lox 66 or lox 71. Further preferred recombinase recognition sites besides the already mentioned Cre/Lox and Flp/Frt sites are Dre/Rox, Vika/vox, VCre/VloxP, SCre/SloxP, λ-Int/attP, R/RRT, Kw/KwRT, Kd/KdRT, B2/B2RT, B3/B3RT (Meinke et al., Chem. Rev. 2016, 116, 20, 12785-12820, incorporated herein by reference). Dre/Rox and Vika/vox are particularly useful in mammalian cells or systems.
Especially preferred are a loxP or FRT site, more preferred a loxP site, even more preferred a loxP site comprising the nucleic acid sequence ATAACTTCGTATAAGGTATCCTATACGAAGTTAT (SEQ ID NO: 17); a lox 66 site or a lox 71 site, a FRP site, a FRT site, especially preferred a FRT site comprising the nucleic acid sequence GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC (SEQ ID NO: 18).
SEQ ID NO: 17 is a Lox2372 site. Another preferred loxP site is LoxP-wt (ATAACTTCGTATAGCATACATTATACGAAGTTAT, SEQ ID NO: 19). FRT sites can be selected from FRT-wt, F1-FRT, F3-FRT and others. Of these, FRT-wt worked best and had no discernible hypomorphic effect. FRT-wt is particularly preferred.
In some embodiments, the genetic element comprises two or more splice branch points, preferably wherein two splice branch points are at a distance of 1 to 10 nucleotides in length to each other. In other embodiments, the genetic element comprises only one branch point.
All of these embodiments can of course be combined, e.g. as shown in the examples. For example, the inventive genetic element comprises the sequences, in 5′ to 3′ direction: a splice donor site, a first recombinase recognition site, a splice branch point comprising an A nucleotide, preferably in the sequence PuAPy, a second recombinase recognition site, a splice acceptor site comprising an A nucleotide, preferably in the sequence AG, wherein the A of the splice branch point is at a distance of 11 to 57 nucleotides in length from the A of the splice acceptor site. Distances are nucleotide between these A's not counting the A's themselves. Said distance may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 52, 53, 54, 55 or 56 nucleotides in length. Pu are purine bases and Py are pyrimidine bases as mentioned above. In a preferred combination, the inventive genetic element comprises the sequences, in 5′ to 3′ direction: a splice donor site comprising the sequence GTPuAG, a first recombinase recognition site, a splice branch point comprising the sequence CTPuAPy, a second recombinase recognition site, a splice acceptor site comprising the sequence AG, wherein the A in bold of the splice branch point is at a distance of 11 to 57 nucleotides in length from the A in bold of the splice acceptor site. Distances are nucleotide between these A's not counting the A's themselves. Said distance may be as given above, i.e. any number from 11 to 57 in preferred embodiments. Pu are purine bases and Py are pyrimidine bases as mentioned above.
Preferred examples of the genetic element of the invention are provided in SEQ ID NOS: 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 23, 25, 27, 29, 31, 33, 35 or part thereof from the splice donor to the splice acceptor, including these sequence elements, as shown in bold in the sequences as depicted in examples 1 and 5. The invention also provides a genetic element comprising a sequence of any one of SEQ ID NOS: 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 23, 25, 27, 29, 31, 33, 35 or part thereof from the splice donor to the splice acceptor, including these sequence elements, as shown in bold in the sequences as depicted in examples 1 and 5, or a sequence with at least 50%, preferably least 60%, least 70%, least 80%, least 90%, least 95%, least 98%, sequence identity to any one of SEQ ID NOS: 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 23, 25, 27, 29, 31, 33, 35 or part thereof from the splice donor to the splice acceptor, including these sequence elements, as shown in bold in the sequences as depicted in examples 1 and 5. SEQ ID NO: 2 and any sequence variant with the given identities are particularly preferred among these sequences.
The invention further relates to a genetic vector comprising the genetic element of the invention. The genetic vector can be an expression or integration vector, a single strand DNA oligo template, a double strand DNA template, a transposon, or a viral vector, preferably an adenoviral, adeno-associated viral, retro- or lentiviral vector, comprising the genetic element of the invention. The vector preferably comprises DNA or RNA. The vector may be used to transfect or transform a cell. The genetic element may be integrated into the genome of the cell.
The genetic element can be introduced as an intron into a gene of a cell and/or it is possible to introduce a gene or a genetic coding element with a genetic element of the invention as an intron, to a cell. Accordingly, the invention further provides a method of providing a cell with a conditionally deactivatable gene, comprising providing a cell, introducing the genetic element of the invention into an exon of a gene in a cell, or introducing a gene with the genetic element of the invention into a cell. Such a genetic element can be introduced using a vector of the invention. The genetic element can be inserted into an exon of the gene or it can be used to replace an intron in a gene with the inventive genetic element. It is also possible to modify an endogenous intron of a gene to resemble a genetic element of the invention so that the result is a genetic element of the invention in the intron. In essence, a gene can be created with the genetic element of the invention flanked by exon sequences. These are all options for introducing an intron into a gene. In preferred embodiments, genetic element of the invention is inserted into a sequence containing a splice junction consensus sequence selected (A/C)-A-G-(A/G) (Burset et al. Nucleic Acids Res. 28, 4364-4375 (2000)) or (A/G/C)-(A/T/G)-G(A/T/G/C) (Ma et al. PLOS One 10, 1-12 (2015)), wherein nucleotides in brackets are alternatives at its position.
This allows the controlled expression of a gene that comprises the genetic element of the invention as an intron. The controlled gene expression of the invention is possible by introducing the genetic element of the invention into an exon which is still allowing an unaltered gene expression of the target gene since the genetic element of the invention is spliced. Following recombinase mediated intron function inactivation at a desired point in time the gene becomes inoperative due to lack of intron splicing. E.g. the non-spliced intron leads to transcription or translation stop or to the expression of a non-functional gene.
The inventive method may comprise introducing into a cell a sequence-specific nuclease that cleaves a sequence within an exon of the target gene thereby introducing the genetic element of the invention into the target gene. Preferably the introduction into an exon of a gene comprises CRISPR-Cas, preferably CRISPR-Cas9, mediated insertion. Further methods for the introduction are with a Zinc finger nuclease, TALEN, Cpf1, and other Cas9-related systems. The genetic element can be introduced by homology directed repair (HDR) or non-homologous end joining (NHEJ).
CRISPR gene editing has facilitated the investigation of gene functions by precise gene knockouts or knock-ins. Upon gRNA-directed double-strand breaks (DSBs), the preferred repair pathway, non-homologous end-joining (NHEJ), often leads to random insertions or deletions (INDELS), where out-of-frame mutations can lead to partial or complete gene loss of function. Using a DNA template, DSBs can also be repaired via homology directed repair (HDR), which allows precise knock-ins of various sequences to target loci. Due to the versatility of CRISPR/Cas9 system and wide applicability, it has been utilized in numerous cell lines and lab organisms spanning the entire biological and biomedical research fields (Pickar-Oliver, A. & Gersbach, C., Nature Reviews Molecular Cell Biology 20, 490-507 (2019)). Accordingly, CRISPR gene editing can be used to introduce the genetic element of the invention into a target gene.
The invention further provides a cell comprising a gene with two or more exons and at least one intron, wherein the intron comprises the genetic element of the invention. The intron may be located between two exons. The cell may be obtainable by the inventive method. In preferred embodiments the cell is a eukaryotic cell, in particular preferred a vertebrate cell, such as a mammalian cell, a fish cell, a bird cell, an amphibian cell, a marsupial cell, a reptile cell; or an invertebrate, such as arthropod, a mollusk, an annelid or a cnidarian. Also possible are insect cells, plant cells, bacterial cells, fungi cells etc. Preferably the cells are selected from a differentiated cell or a pluripotent cell. In some embodiments, human totipotent cells are excluded. In other embodiments, any totipotent cell is excluded. The cell may be from a cell line, a cell culture cell or an isolated cell originating from but removed from a non-human animal or a human. The genetic element of the invention can be used as intron in a gene in pluripotent stem cells, cell lines, organoids, primary cells; such as human pluripotent stem cells, human cell lines, human organoids, primary human cells; or from any of the above organisms.
Conditional gene inactivation with the genetic element of the invention can be used to reduce viability or survivability of cells, such as by placing it in essential genes. Conditional gene inactivation can be used in removing transplanted human cells when, e.g. they are no longer needed in a cell therapy. For example, one can use the genetic element in CAR-T cells so that all treated CAR-T cells can be killed when the CAR-T cells are no longer needed or become detrimental for a recipient.
Further provided is a non-human animal comprising one or more cells of the invention. The non-human animal may be any organism mentioned above, e.g. a vertebrate, such as a mammal, a fish, a bird, an amphibian, a marsupial, a reptile. The cells of the invention may be provided or introduced to a human or a nonhuman animal.
The present invention further relates to a method of inactivating expression of a functional gene in a cell or non-human animal, comprising providing a cell of the invention or a nonhuman animal of the invention and activating recombination at the recombinase recognition sites in the cell of the invention or in a cell in the non-human animal of the invention. Activation of the recombinase recognition sites leads to an excision of the sequence between the recombinase recognition sites and thereby removal of the splice branch point, thereby removing the potential to splice the genetic element. For this, in all embodiments of the invention, the recombinase recognition sites should be in the same orientation. By removing the splicing potential, the gene containing the inventive genetic element will no longer be expressed properly as the genetic element without splicing leads to a malfunctioning gene, e.g. by containing a non-functional or disruptive sequence to the expressed gene, or an abrogated expression, such as by a premature termination. For a premature termination, the inventive genetic element preferably contains one or more stop codons. Preferably there is at least one stop codon in each reading frame, e.g. as described in WO 2018/096356 A1.
The method may comprise the step of introducing or activating a recombinase in the cell thereby excising or disrupting the branch point and abrogating splicing of the genetic element of the invention with the SCON. The recombinase acts on the first and second recombinase sites and excises sequence in between. Activation or introduction may comprise initiating expression of the recombinase or providing an activator for it. For example, recombinases can be expressed with a receptor that mediates activation. An example thereof is CreER, a Cre recombinase with an estrogen receptor (ER) that leads to the activation of the Cre part upon ER ligand binding, such as binding of a ligand like tamoxifen or 4-hydroxytamoxifen. A preferred type of CreER is CreERt2.
The inventive genetic element can be used for targeting an endogenous gene in a cell. Alternatively, the overexpression of the inventive genetic element can be used to investigate the activity of a promoter, a transcription factor, a transcription cofactor, splicing factors, and regulation of splicing. For example, it is possible to study a reduced expression, e.g. a knock-out, or overexpression of such factors to screen for depleted or elevated expression, compared without these factors. For such studies, the genetic element of the invention may be in an intron of a reporter gene such as a fluorescent protein, like GFP, or an antibiotic resistance gene.
The invention provides a method of investigating the function of a gene, comprising inactivating a functional gene according to the method of the invention and comparing the inactivated gene's effect in the cell or non-human animal to a cell or non-human animal without inactivation of the gene or to a cell or non-human animal without the genetic element of the invention. The comparison to a state without the genetic element of the invention or to the state without inactivation serves as a control to identify differences caused by the gene under investigation that comprises the genetic element of the invention. The inactivation of a functional gene may lead to an observable effect, such as altered cell function, viability of gene expression. As mentioned above, the functional gene may be a reporter gene with an expected expression pattern difference before and after inactivation, wherein other activities of the cell affecting the reporter gene, such as splicing or promoter functions, are investigated.
Further provided is a kit suitable for integrating an intron into a target gene comprising a genetic element of the invention and a Cas encoding nucleic acid and/or a Cas protein, preferably further comprising a CRISPR-Cas guide nucleic acid targeting an exon in the target gene. The Cas protein is preferably Cas9; the Cas encoding nucleic acid is preferably a Cas9 encoding nucleic acid. The Cas enzyme may be Cas1, Cas2, Cas3, Cas9, dCas9, Cas10 or Cas12a. The CRISPR/Cas method typically comprises the use of single guide RNA (sgRNA) that comprises both the crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as a single construct. The crRNA is also referred to as guideRNA for containing the DNA guiding sequence. The guideRNA may be specific for a gene wherein the inventive genetic element may be inserted into. The tracrRNA and the crRNA can be linked to form a single molecule, i.e. the single guide RNA (sgRNA). tracrRNA and crRNA hybridize in a complementary region. This complementary region can be used for the linkage and may form, together with a linkage, a stem-loop, called the crRNA: tracrRNA stem loop herein. Since this region in most cases mediates binding to a Cas protein, it may also be referred to as Cas binding element. Site-specific cleavage occurs at locations determined by both base-pairing complementarity between the crRNA and the target protospacer DNA, and a short motif [referred to as the protospacer adjacent motif (PAM)] juxtaposed to the complementary region in the target DNA. The target DNA may be in any DNA molecule that should be modified. It may be of a gene that shall be modified. A typical use of the CRISPR/Cas system is to introduce mutations or modifications that allows the insertion of the genetic element of the invention. The design of sgRNAs is by now conventional, as reviewed e.g. by Ciu et al. (Interdisciplinary Sciences Computational Life Sciences 2018, DOI: 10.1007/s12539-018-0298-z) or Hwang et al. (BMC Bioinformatics 19, 2018: 542). Many tools exist that can be used according to the invention to generate a sgRNA sequence targeting a gene of interest.
The kit may comprise a guideRNA targeting a gene of interest. The kit may also comprise a tracrRNA suitable for a CRISPR-Cas method. The guideRNA and tracrRNA may be in a single RNA molecule, as in a sgRNA. Alternatively or in addition, the kit may comprise a nucleic acid encoding any of these RNA (any of guideRNA, tracrRNA and/or sgRNA).
The kit can be used for various uses, such for zygote pronuclear injection, zygote cytoplasmic transfection, or for 2-cell cytoplasmic transfection.
The invention further provides a method of introducing an intron sequence into an exon or between two exons of a gene, comprising the steps of selecting the exon or one of the two exons, respectively, which is positioned within the first 50% base pairs (bp) of a protein coding-sequence of the gene; and the intron is inserted into an intron insertion site containing either a stringent splice junction consensus sequence or a flexible splice junction consensus sequence; and wherein after the introduction of the intron the intron is separating exons on the intron's 5′ and 3′ sides with the exons being each at least 60 bp in length.
In preferred embodiments, the stringent splice junction consensus sequence is A/C)-A-G-(A/G) (Burset et al. Nucleic Acids Res. 28, 4364-4375 (2000). In further preferred embodiments, the flexible splice junction consensus sequence comprised the sequence (A/G/C)-(A/T/G)-G-(A/T/G/C) (Ma et al. PLOS One 10, 1-12 (2015)).
Preferably, an exon that is to be spit by introducing the intron into the exon is at least and the exon is at least 120 bp in length.
The protein coding-sequence of the gene can be of a genome, e.g. be located on a chromosome. The gene can be of a cell or organism as described above for the genetic element of the invention (SCON), such as in a cell from a cell line, a cell culture cell or an isolated cell originating from but removed from a non-human ani-mal or a human.
The intron can be used to replace an existing intron, e.g. as embodiment of introducing the intron sequence between two exons of a gene, or introduced into an exon that in the target gene would not have an intron at the selected site of intron insertion.
The intron may be a conditional intron that can have an intron function during splicing under selectable or controllable conditions, i.e. the intron can be inactivated or activated. For such embodiments, the functional intron may have a splice donor site, a splice branch point, and a splice acceptor site, functionally positioned to act as an intron during splicing, e.g. being excised from a transcript. The functional position may be disrupted by recombinase recognition sites, that may change the functional positioning by action of a recombinase. A preferred intron is a genetic element of the invention (SCON) as described above. The SCON is an example of an intron with splicing function that can be deactivated upon recombinase action, turning the intron non-functional. E.g. the intron may comprise a splice donor site, a first recombinase recognition site, a splice branch point, a second recombinase recognition site, a splice acceptor site. A pair of recombinase recognition sites, e.g. a first and a second recombinase recognition sites, may be used to alter the availability of a site necessary for splice function, such as the splice donor site, a splice branch point, and/or a splice acceptor site, for splicing. The alteration may be a functional removal or functional activation of splicing function. A functional removal may be a physical removal of one or more of the sites, such as an excision, by the recombinase. Another functional alteration may turn an inactive site active, such as by removing inhibitory RNA structures by the recombinase. As mentioned above, a recombinase may remove the part or site flanked by a pair of recombinase recognition sites.
Throughout the present disclosure, the articles “a”, “an”, and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refer to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by e.g. ±10%.
As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The “comprising” expressions when used on an element in combination with a numerical range of a certain value of that element means that the element is limited to that range while “comprising” still relates to the optional presence of other elements. E.g. the element with a range may be subject to an implicit proviso excluding the presence of that element in an amount outside of that range. As used herein, the phrase “consisting essentially of” requires the specified integer (s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the closed term “consisting” is used to indicate the presence of the recited elements only.
The present invention is further described by the following figures and examples, without necessarily being limited to these embodiments of the invention.
All animal experiments were performed according to guidelines of the Austrian Animal Experiments Acts, and with valid project licenses which were approved by the Austrian Federal Ministry of Education, Science and Research and monitored by the institutional IMBA ethics and Biosafety department.
A Ctnnb1-SCON (Ctnnb1sc) conditional KO mouse was generated via 2-cell stage embryo injection. To prepare the CRISPR injection mix, the following component was mixed together in 25 μl with the final concentration in brackets: spCas9 mRNA (100 ng/μl,), spCas9 protein (50 ng/μl), sgRNA (50 ng/μl) and SSODN (20 ng/μl, Genscript). The mixture was spun down in a tabletop centrifuge at 4° C. 13,000 g for 15-20 minutes to prevent clogging of injection needles. Frozen 2-cell stage embryos of C57Bl/6JRj background (JANVIER LABS) were used for the cytoplasmic injection.
Ctnnb1-SCON was crossed with Vil-CreERT2 (JAX, 020282, El Marjou et al. genesis 39, 186-193 (2004)) and bred to obtain either HET (Vil-CreERT2; Ctnnb1+/sc) or HOM (Vil-CreERT2; Ctnnb1sc/sc) mice. Tamoxifen (Sigma, T5648) dissolved in corn oil (Sigma, C8267) or corn oil only was injected intraperitoneally into 8-12 weeks old mice at final concentration of 3 mg Tamoxifen per 20 g body weight for CreER activation. Controlled mice received matched volume of corn oil. Uninduced, Day 3 or Day 5 mice were euthanized by cervical dislocation, and the intestines were harvested. The intestines were immediately cleaned with 1×PBS and flushed gently with 10% formalin solution (Sigma, HT501128), and fixed as ‘swiss rolls’ for 24 hours at room temperature. Fixed intestines were washed three times with 1×PBS, with 2-3 hours between each wash, before further processing.
The toe clips or ear notches from the offspring were lysed in 30 μl of DirectPCR Lysis Reagent (Viagen) with 1 μl of proteinase-K (20 mg/ml; Promega, MC5005) at 55° C. overnight. The resulted mixture was diluted with 270 μl of nuclease-free water and spun down for at least 5 minutes in a tabletop centrifuge at 13, 000 g. Then, 2-3 μl of the clear part of the solution was used for PCR reaction, with either Gotaq (Promega, M7808) or LongAmp 2× (NEB, M0287S). To check the sequences of genomic DNA, PCR bands at expected sizes were purified with a purification column.
eGFP-SCON/eGFP-DECAI Constructs
The eGFP-SCON and eGFP-DECAI cassettes, where SCON or DECAI is inserted in the middle of eGFP, were synthesized and ordered from Genscript, and subsequently cloned into pcDNA4TO construct with BamHI (R0136S, NEB) and XhoI (R0146S, NEB) via ligation with T4 ligase (M0202S, NEB). The vectors were recombined with Cre-expressing bacteria (A111, Gene bridges) to obtain the recombined forms. The correct clones were confirmed with restriction digest SalI (R0138S, NEB) and Sanger sequencing.
SCON A-Stretch Variants Inserted in the eGFP cDNA
eGFP CDNA containing the SapI recognition sites at the selection intron insertion site, where the intron splice donor and acceptor was first cloned in the pcDNA4TO construct with BamHI and XhoI. Different SCON variant fragments containing the respective complementary ends were then inserted in the eGFP with SapI (R0569S, NEB) and T4 ligase for 20 cycles of 2 minutes at 37° C. and 5 minutes at 16° C. shuffling reaction. The mixture was transformed into Escherichia coli and DNA was extracted from individual colonies checked with restriction digests and Sanger sequencing.
Human embryonic kidney (Hek) 293T cell culture was cultured in DMEM with high glucose with 10% fetal bovine serum (FBS, Sigma), 1% penicillin-streptomycin (P/S; Sigma, P0781) and 1% L-glutamine (L-glut; Gibco, 25030024).
Mouse ESCs (AN3-12) were cultured, as previously reported (Elling et al. Nature 550, 114-118 (2017)), in DMEM with high glucose (Sigma, D1152), 10% FBS (Sigma), 1% P/S, 1% L-glut, 1% NEAA (Sigma, M7145), 1% sodium pyruvate (Sigma, S8636), 0.1 mM 2-mercaptoethanol (Sigma, M7522), 7.5 μl of mouse LIF (stock concentration: 2 mg/ml).
The following cell lines were cultured with medium supplemented with 10% FBS and 1% P/S, the corresponding basal medium are indicated in brackets: C6 (ATCC, CCL-107; DMEM-F12 (Gibco, 31330038)), PK15 (Elabscience Biotechnology, EP-CL-0187; Minimal essential medium (Gibco, 11095080), LLC-MK2 (Elabscience Biotechnology, ELSEP-CL-0141-1; RPMI-1640 (Sigma, R8758)), Vero (ATCC, CCL-81; DMEM-High glucose (Sigma, D1152)).
500,000-750, 000 Cells were seeded in 6-well plates and left to attach and grow overnight. 2.5 μg of DNA (1 ug of mCherry-expressing plasmid (Addgene, 72264), and 1.5 μg of pcDNA4TO-eGFP, -eG-SCON-FP, -eG-DECAI-FP or recombined forms of eG-SCON-FP or eG-DECAI-FP) was mixed with 8 μl of polyethyleneimine (1 mg/ml, 23966) and incubated at room temperature for at least 15 minutes before being added dropwise to the cells. Culture media was exchanged 8-10 hours after transfection. 36 hours after transfection, cells were examined under the EVOS M7000 microscope (Thermo Scientific) with the brightfield, GFP and TexasRed filters. 36-48 hours after transfection, cells were dissociated into single cells for flow cytometry analysis, with a BD-LSRFor-tessa flow cytometer (BD). Data from the flow cytometry experiments were analyzed in FlowJo software (BD).
Crypts were isolated from the proximal part of the small intestine as reported previously (Sato et al. Nature 459, 262-265 (2009)) and embedded in 15 μl BME-R1 (R&D Systems, 3433010R1) droplets in a 48-well plate (Sigma, CLS3548-100EA). Organoids were established in WENR+Nic medium consisting of advanced Dulbecco's modified Eagle's medium/F12 (DMEM-F12; Gibco, 12634028) supplemented with pen/strep (100×; Sigma, P0781), 10 mM Hepes (Gibco, 15630056), Glutamax (100×; Gibco, 35050061), B27 (50×; Life Technologies, 17504044), Wnt3 conditioned medium (Wnt3a L-cells, 50% of final volume), 50 ng/ml recombinant mouse epidermal growth factor (EGF; Gibco, PMG8041), 100 ng/ml recombinant murine Noggin (PeproTech, 250-38), R-spondin-1 conditioned medium (HA-R-Spondin1-Fc 293T cells, 10% of final volume) and 10 mM nicotinamide (MilliporeSigma, N0636). For the first week of culture, primocin (InvivoGen, ant-pm-05) and ROCK-inhibitor/Y27632 (Sigma, Y0503) were supplemented to prevent microbial contamination and apoptosis, respectively. After the first passage, established organoids were converted to ENR budding organoid culture. Organoids were passaged with mechanical dissociation and diluted in 1:6 ratio.
Budding organoids with passage 3 or higher were passaged with mechanical dissociation and seeded in BME droplets. Media containing vehicle (ethanol) or 500 nM 4-hydroxytamoxifen (Sigma, H7904) were added after BME polymerized. 8 hours after, media was exchanged back to ENR and replenished every two days.
Samples were processed using standard tissue protocol on Automatic Tissue Processor Donatello (Diapath). Samples were embedded in paraffin and were cut into 2 μm sections onto glass slides. Hematoxylin and Eosin staining was done according to the standard protocol and using Gemini AS stainer (Thermo Scientific). For the immunohistochemistry following antibody staining, the following antibodies were used: Rabbit anti-Ki67 (1:200; 2 hours at room temperature; Abcam, Ab16667), Rabbit anti-β-catenin (1:300; 1 hour at room temperature; Abcam, ab32572). For signal detection, a two-step Rabbit polymer system HRP conjugated (DCS, PD000P0L-K) was used. Stained slides were imaged with the 40× objective using the Pannoramic FLASH 250 III scanner (3DHISTECH) and images were cropped using the CaseViewer software.
BME droplets containing organoids were carefully collected into 1.5 ml tubes and spun down in a tabletop centrifuge at 600 g for 5 minutes. The supernatant and visible fraction of attached BME were removed. The pellet was resuspended in 4% paraformaldehyde and fixed at room temperature for 15-20 minutes. Fixed organoids were washed in 1×PBS for 3 times with 10-15-minute intervals. The organoids were blocked and permeabilized in a solution containing 5% DMSO, 0.5% Triton-X-100 (Sigma, T8787) and 2% normal donkey serum (Sigma, D9663) for one hour at 4° C. Then, the samples were stained overnight with Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S), ATTO 488-conjugated phalloidin (1:300; Sigma, 49409-10NMOL). The samples were washed three times with 1×PBS and during the last wash, the samples were incubated with 2 μg/ml DAPI and mounted onto coverslips in a solution containing 60% glycerol and 2.5M fructose (Dekkers et al. Nature Protocols 14, 1756-1771 (2019)). The imaging was done with a multiphoton SP8 confocal microscope (Leica).
In order to construct databases for SCON insert sites, we used genomic information, about sequence, exon, coding region, and gene type, derived from Ensembl Biomart, build 102 (www.ensembl.org/, Yates et al., Nucleic Acids Research, 2019, Ensembl 2020) for mouse (M. musculus), rat (R. norvegicus), macaque (M. mulatta), marmoset (C. jacchus), and medaka (O. latipes). In order to map canonical transcripts to genes, we derived ‘PRINCIPAL: 1’ from APPRIS database for mouse and rat with Ensembl, build 102 (appris-tools.org, Rodriguez, J. M. et al. Nucleic Acids Res. 46, D213-D217 (2018)) and we regard the longest transcript as canonical transcript for other species. The quality features including GC-content, self-complementarity, and mismatch scores for each the candidate sites are mapped by same approach as CHOPCHOP (chopchop.cbu.uib.no, Labun, K. et al. Nucleic Acids Res. 47, W171-W174 (2019).
This example shows the generation and use of a Short Conditional intrON (SCON) cassette that shows no hypomorphic effect in various vertebrate species and is compatible for targeting via one-step zygote microinjections.
The generation of conditional allele using CRISPR technology is still challenging. As best mode a SCON of 189 bp in size is used to enable a rapid generation of conditional allele with one-step zygote injection. SCON has conditional intronic function in various vertebrate species and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.
The SCON shown here for illustration purposes is a modified intron derived from the first intron (130 bp) of human HBB (Hemoglobin subunit beta) gene. In complete form, this SCON is 189 bp long, consisting of, from the 5′ to 3′ end, a splice donor, LoxP recombination site 1, a branch point, LoxP recombination site 2, polypyrimidine tract, and a splice acceptor. By design, it has a similar sequence architecture to the previously introduced conditional intronic system—DECAI (201 bp, Guzzardo et al. Scientific Reports 7, (2017)), which however showed a hypomorphic effect at the level of protein expression.
To optimize conditional intron function, several features were implemented into SCON, including: 1) the optimized length between the putative branch point and the splice acceptor is kept at 45 bp to allow efficient splicing to take place, 2) 100 bp distance was used between the two LoxP sites for an efficient Cre-LoxP recombination, and 3) other miscellaneous changes were incorporated for optimal splice donor, acceptor and pyrimidine tract sequences. Upon recombination, SCON is reduced to 55 bp, of which all three reading frames contain translational stop codons within the remaining LoxP sequence such that gene loss of function occurs via premature translational termination.
TATAAGGTATCCTATACGAAGTTATTCTCTCTGCCTATTGGGGTTACAAG
CGAAGTTATTTTCCCTCCCTCAGGacAGAAGAGCGGGCAACTTGCCCCAT
Firstly, we validated the functionality of SCON in an eGFP overexpression construct. We co-transfected mCherry cassette (serving as transfection control) with a cassette containing either the intact eGFP, eG-SCON-FP or already Cre-recombined eG-SCON-FP (
To understand better the functionality of different parts within the SCON cassette, we designed a series of “A-stretch variants” where 6-10 nt within SCON is converted into adenine (
AAAAAAAATAACTTCGTATAAGGTATCCTATACGAAGTTATTCTCTCTGCCTATTGGGGTTACA
AAAAAAATTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCT
AAAAGGCACTGACATAACTTCGTATAAGGTATCCTATACGAAGTTATTTTCCCTCCCTCAG
Out of the 13 different variants, we found that four variants had either hypomorphic or complete reduction of eGFP fluorescence when inserted as an intron (
Next, we tested whether SCON can be efficiently recombined in mammalian cells. We cloned the eG-SCON-FP construct into a PiggyBac transposon backbone, and generated a clonal mouse embryonic stem (ES) cell line with constitutive eGFP expression containing SCON. By co-transfecting Cre-expressing plasmid with mCherry cassette into eG-SCON-FP-expressing ES cells, we observed an efficient reduction of eGFP level within the mCherry+ population whereas mock control (transfected only with mCherry cassette) maintained high levels of eGFP (
cKO alleles have been widely used in mice for decades thanks to the robust set up with germline competent ES cells (Mulas et al. Development 146, dev173146 (2019)), the endeavor of International Knockout Mouse Consortium, and also recently via the advent of CRISPR technology (Quadros et al. Genome Biology 18, (2017)). However, for many other vertebrate species such as zebrafish, rat, porcine, bovine and non-human primate models, the use of cKO approach has been limited largely due to the lack of reliable germline competent ES cells. We sought to test whether SCON would be a suitable cKO strategy for other species. Therefore, we made use of cell lines of different species, including C6 (Rattus norvegicus), PK15 (Sus scrofa), LLC-MK2 (Macca mulatta) and Vero (Cercopithecus aethiops) and transfected them with overexpression constructs of eGFP, eG-SCON-FP or eG-DECAI-FP and the corresponding Cre-recombined forms. In line with the results in HEK293T and mES cells, SCON intron did not show any discernable hypomorphic effect in all tested cell lines (
As previous in vitro overexpression-based system highlighted the desired features of SCON for cKO approaches, we sought to test whether SCON would also work well in targeting endogenous genes. Therefore, we chose to generate SCON cKO Ctnnb1 allele (
To verify the conditional functionality of Ctnnb1sc allele, we utilized the Villin-CreERT2 (Vil-CreERT2) for intestinal epithelium-specific Cre recombination. We first isolated crypts from the duodenum of HOM (Vil-CreERT2; Ctnnb1sc/sc) and wildtype (Ctnnb1+/+) mice to establish adult stem cell (AdSC)-based intestinal organoids. Then, transient 4-Hydroxytamoxifen (4-OHT) treatment for 8 hours was carried out in budding organoid culture (with Egf, Noggin and R-spondin). Both 4-OHT treated and untreated wildtype organoids and untreated HOM organoids grew normally (
To directly verify the functionality in vivo, we injected 3 mg Tamoxifen (TAM) per 20 g body weight into both HOM (Vil-CreERT2; Ctnnb1sc/sc) and HET (Vil-CreER T2; Ctnnb1+/sc) mice, and harvested the intestines on day 3 and 5. Control samples showed normal crypt-villus axis, detectable membrane-bound beta-catenin, and Ki67+ proliferating zone in the bottom of the crypts where stem and progenitor cells are located (
To systematically estimate optimal sites for easy access for SCON integration, we carried out bioinformatic analysis to screen for insertion sites in mouse, rat, macaque, marmoset and medaka genomes (
To test whether SCON alleles could be generated in multiple mice genes without observable hypermorphic effect, we targeted a total of seven genes, including Ctnnb1, with SCON. Of these, three genes were targeted with the LoxP version and four with the Frt version. Three genes, Ctnnb1 (Huelsken et al., J. Cell Biol. 148, 567-578 (2000)), Sox2 (Avilion et al., Genes Dev. 17, 126-140 (2003)), Sav1 (Lee et al., EMBO J. 27, 1231-1242 (2008)), are known to cause developmental lethality upon whole body or organ-specific knockout. Two genes, Mlh1 (Edelmann et al., Cell. 85, 1125-1134 (1996)) and Usp42 (White et al., Cell. 154, 452 (2013)), were reported to cause sterility upon whole body knockout. Lastly, Lpar2 knockout mice are viable and fertile but show phenotypes in signaling deficits in response to lysophosphatidic acid (Contos et al., Mol. Cell. Biol. 22, 6921-6929 (2002)), and Ace2 knockout mice are also viable and fertile but are more susceptible to tissue damages such as in the heart (Crackower et al., Nature. 417, 822-828 (2002)) and lung (Imai et al., Nature. 436, 112-116 (2005)).
SCON insertions were generated by injecting CRISPR ribonucleoprotein (RNP) with ca. 300 bp-long, company-synthesized, single-stranded deoxynucleotides (ssODNs) consisting of SCON with short homology arms) into the cytoplasm of the developing either 2-cell (only Ctnnb1) or 1-cell stage mouse embryos (Gu et al. Nature Biotechnology 36, 632-637 (2018)). SCON designs for cloning with 5′ and 3′ additions (similar to SEQ ID NO: 3) are provided in the following:
highlighted elements from 5′ to 3′: bold: splice donor; underlined: loxP site; 2× bold: 2× branch point; underlined: italic: polypyrimidine tract; loxP site; bold: splice acceptor
Ctnnb1-gRNA used for CRISPR-Cas9 targeting: TACATCATTTGTATTCTGCA (SEQ ID NO: 24) The Ctnnb1-gRNA sequence recognizes position 51-55 and 245-259 in the reverse orientation and the PAM site is in position 48-50.
TTCCTATTCtctagaaaGtATAGGAACTTC
TTTCCCTCCCTC
AGGAGCAC
highlighted elements from 5′ to 3′: bold: splice donor; underlined: Frt site; 2× bold: 2× branch point; underlined: Frt site; italic: polypyrimidine tract; bold: splice acceptor
Sox2-gRNA used for CRISPR-Cas9 targeting: TCTGCACATGAAGGAGCACC (SEQ ID NO: 26) The Sox2-gRNA sequence recognizes position 43-55 and 245-251 in the forward orientation and the PAM site is in position 252-254.
CTTCGTATAAGGTATCCTATACGAAGTTAT
TTTCCCTCCCTC
AGGCTCTC
highlighted elements from 5′ to 3′: bold: splice donor; underlined: loxP site; 2× bold: 2× branch point; underlined: loxP site; italic: polypyrimidine tract; bold: splice acceptor
Lpar2-gRNA used for CRISPR-Cas9 targeting: ATGGAGAGCCTGGCAGTGCG (SEQ ID NO: 28) The Lpar2-gRNA sequence recognizes position 45-55 and 245-253 in the reverse orientation and the PAM site is in position 42-44.
TTCCTATTCtctagaaaGtATAGGAACTTC
TTTCCCTCCCTC
AGATGGTC
highlighted elements from 5′ to 3′: bold: splice donor; underlined: Frt site; 2× bold: 2× branch point; underlined: Frt site; italic: polypyrimidine tract; bold: splice acceptor
Mlh1-gRNA used for CRISPR-Cas9 targeting: CAAGGTCTACGCTTACCAGA (SEQ ID NO: 30) The Mlh1-gRNA sequence recognizes position 37-55 and 245 in the forward orientation and the PAM site is in position 246-248.
TTCCTATTCtctagaaaGtATAGGAACTTC
TTTCCCTCCCTC
AGACTCCG
highlighted elements from 5′ to 3′: bold: splice donor; underlined: Frt site; 2× bold: 2× branch point; underlined: Frt site; italic: polypyrimidine tract; bold: splice acceptor
Ace2-gRNA used for CRISPR-Cas9 targeting: GACGCTTGATGATCGGAGTC (SEQ ID NO: 32) The Ace2-gRNA sequence recognizes position 55 and 245-263 in the reverse orientation and the PAM site is in position 52-54.
CTTCGTATAAGGTATCCTATACGAAGTTAT
TTTCCCTCCCTC
AGAATTTG
highlighted elements from 5′ to 3′: bold: splice donor; underlined: loxP site; 2× bold: 2× branch point; underlined: loxP site; italic: polypyrimidine tract; bold: splice acceptor
Usp42-gRNA used for CRISPR-Cas9 targeting: GGCGCTGGGCTCCAGAATTT (SEQ ID NO: 34) The Usp42-gRNA sequence recognizes position 41-55 and 245-249 in the forward orientation and the PAM site is in position 250-252.
TTCCTATTCtctagaaaGtATAGGAACTTC
TTTCCCTCCCTC
AGAGTTTC
highlighted elements from 5′ to 3′: bold: splice donor; underlined: Frt site; 2× bold: 2× branch point; underlined: Frt site; italic: polypyrimidine tract; bold: splice acceptor
Sav1-gRNA used for CRISPR-Cas9 targeting: TTGCAGTTCTCAGGAAACTC (SEQ ID NO: 36) The Sav1-gRNA sequence recognizes position 55 and 245-263 in the reverse orientation and the PAM site is in position 52-54.
Here, in addition to the previous examples, in addition to Ctnnb1, six additional conditional KO mice were generated via 1-cell stage embryo injection. To prepare the CRISPR injection mix, the following components were mixed together in 25 μl with the final concentration in brackets: spCas9 mRNA (100 ng/μl,), spCas9 protein (50 ng/μl), sgRNA (50 ng/μl) and ssODN (20 ng/μl, Genscript). The mixture was spun down in a tabletop centrifuge at 4° C., 13,000×g for 15-20 minutes to prevent clogging of injection needles. Frozen 1-cell stage embryos of C57Bl/6JRj background (JANVIER LABS, France) were used for the pronuclear injection.
†SCON targeted to genes important for fertility;
§SCON targeted to non-lethal genes.
As seen in Table 1, Homo or Hemizygous (for genes on X or Y chromosomes) founders were identified for all lines except for Ctnnb1 and Sox2. The homozygous insertion of Sav1 demonstrates that the SCON allele does not display hypomorphic effects, and such insertions can be easily obtained in a single step process.
Homozygous SCON alleles of Ctnnb1 and Sox2 were subsequently obtained by crossing (
In summary, SCON-mediated cKO approach renders the complicated generation of conditional allele into a simple CRISPR-mediated short sequence knock-in of e.g. 189 bp intronic sequence in the optimized SCON variant with one-step zygote injection. The intron is neutral on expression levels before Cre-mediated inactivation. This novel strategy opens an exciting possibility of applying the same strategy to other vertebrate models ranging from fish to non-human primates. Moreover, the LoxP sequences can also be replaced by other recombination sites (e.g. FRT) for rapid generation of FRT- or other recombinase-based conditional alleles that have not been widely utilized yet. Lastly, the dispensable region between the two LoxP sites or other recombination sites serve as a harboring space for an addition of other genetic elements. The SCON strategy can be a new foundation of cKO approach in biomedical and industrial research that is well suited for animal welfare.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21172761.5 | May 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/062284 | 5/6/2022 | WO |
