Composition for cleaving target DNA and use thereof
阅读说明：本技术 用于切割靶dna 的组合物及其用途 (Composition for cleaving target DNA and use thereof ) 是由 金进秀 曹承于 金素贞 J·M·金 金爽中 于 2013-10-23 设计创作，主要内容包括：本发明涉及真核细胞或生物体中的靶向基因组编辑。更具体地,本发明涉及用于在真核细胞或生物体中切割靶DNA的组合物及其用途,所述组合物包含特异于靶DNA的向导RNA和Cas蛋白质编码核酸或Cas蛋白质。(The present invention relates to targeted genome editing in eukaryotic cells or organisms. More specifically, the present invention relates to a composition comprising a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or Cas protein for cleaving the target DNA in a eukaryotic cell or organism and uses thereof.)
1. A composition for modifying genomic DNA of a eukaryotic cell comprising
A Cas9/RNA complex comprising:
A) a Streptococcus pyogenes (Streptococcus pyogenes) Cas9(s. polynucleotides Cas9) protein or polypeptide linked to a Nuclear Localization Signal (NLS), and
B) a single stranded guide RNA (sgRNA) having CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA),
wherein the crRNA comprises i) an essential portion capable of hybridizing to at least a portion of the tracrRNA, and ii) a portion complementary to the target DNA, and
wherein the essential portion of the crRNA is fused to the portion of the tracrRNA by synthetic additional nucleotides between the essential portion of the crRNA and the portion of the tracrRNA;
wherein the s.pyogenes Cas9 protein or polypeptide is complexed with the single stranded guide rna (sgrna) to form an active endonuclease.
2. The composition of claim 1, wherein the Nuclear Localization Signal (NLS) is located at the terminus of the s.
3. The composition of claim 1, wherein the s.pyogenes Cas9 protein is a recombinant Cas9 protein.
4. The composition of claim 1, wherein the portion of the crRNA of the guide RNA complementary to genomic DNA of the eukaryotic cell comprises 19 to 20 nucleotides.
5. The composition of claim 1, wherein the single stranded guide RNA (sgRNA) is an in vitro transcribed guide RNA.
6. The composition of claim 1, wherein the Cas9/RNA complex does not comprise a DNA molecule.
7. The composition of claim 6, wherein the Cas9/RNA complex is capable of recognizing an NGG trinucleotide in eukaryotic genomic DNA.
8. A method of modifying genomic DNA of a eukaryotic cell, comprising:
introducing the composition into a eukaryotic cell;
wherein the composition comprises a Cas9/RNA complex, the Cas9/RNA complex comprising:
A) a Streptococcus pyogenes (Streptococcus pyogenes) Cas9(s. polynucleotides Cas9) protein or polypeptide linked to a Nuclear Localization Signal (NLS), and
B) a single stranded guide RNA (sgRNA) having CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA),
wherein the crRNA comprises i) an essential portion capable of fusing with a tracrRNA, and ii) a portion complementary to the target DNA; and
wherein the essential portion of the crRNA is fused to the portion of the tracrRNA by synthetic additional nucleotides between the essential portion of the crRNA and the portion of the tracrRNA;
wherein the introduction of the composition into the eukaryotic cell is effected by one of the following methods
Forming a Cas/RNA complex in vitro, then introducing the Cas/RNA complex into a eukaryotic cell, and
preparing the S.pyogenes Cas protein or polypeptide and the synthetic guide RNA in vitro, then introducing the S.pyogenes Cas protein or polypeptide and the synthetic guide RNA into eukaryotic cells, respectively,
wherein the introducing composition introduces site-specific mutations in the genomic DNA of the eukaryotic cell, the mutations being at least one of:
one or more nucleotide insertions in the genomic DNA,
deletion of one or more nucleotides in genomic DNA, and
one or more nucleotide substitutions in the genomic DNA.
9. A genome-modified eukaryotic cell having a mutation introduced in the genome by the composition of any one of claims 1 to 7,
wherein the mutation is at least one of
One or more nucleotide insertions in the genome,
one or more nucleotides in the genome are deleted,
one or more nucleotide insertions and deletions in the genome, and
one or more nucleotide substitutions in the genome;
wherein the genomically modified cell comprises a gene knockout wherein the gene has a mutation in the genome.
10. A composition for modifying genomic DNA of a plant cell, comprising:
(A) Cas/RNA complex comprising
a) A Cas protein or polypeptide linked to a Nuclear Localization Signal (NLS), and
b) a synthetic guide RNA having CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), complexed with the Cas protein or polypeptide in a),
wherein the crRNA comprises i) an essential portion capable of hybridizing to at least a portion of the tracrRNA, and ii) a portion complementary to the genomic DNA;
(B) a complexing buffer for maintaining the Cas protein or polypeptide and the synthetic guide RNA in a complex form; and
(C) a transfection buffer for transfecting a plant cell;
wherein the Cas protein or polypeptide is complexed with a guide RNA (sgRNA) to form an active endonuclease,
wherein the composition is introduced into a double strand break in the genomic DNA of a plant cell in vitro.
11. A method for modifying genomic DNA of a plant cell comprising:
introducing the composition into a plant cell;
wherein the composition comprises
(A) A Cas/RNA complex comprising:
a) a Cas protein or polypeptide linked to a Nuclear Localization Signal (NLS), and
b) a synthetic guide RNA having CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), complexed with the Cas protein or polypeptide in a),
wherein the crRNA comprises i) an essential portion capable of hybridizing to at least a portion of the tracrRNA, and ii) a portion complementary to the genomic DNA;
(B) a complexing buffer for maintaining the Cas protein or polypeptide and the synthetic guide RNA in a complex form; and
(C) a transfection buffer for transfecting a plant cell;
wherein the introduction of the composition into the plant cell is effected by one of the following methods
a) Forming a Cas/RNA complex in vitro, then introducing the Cas/RNA complex into a plant cell, and
b) preparing the Cas protein or polypeptide and the synthetic guide RNA in vitro, and then introducing the Cas protein or polypeptide and the synthetic guide RNA into the plant cell,
wherein the introducing composition introduces a site-specific mutation in the genomic DNA of the plant cell, the mutation being at least one of:
one or more nucleotide insertions in the genomic DNA, one or more nucleotide deletions in the genomic DNA, and
one or more nucleotide substitutions in the genomic DNA.
12. A genome-modified plant cell having a mutation induced in the genome by the composition of claim 10,
wherein the mutation is at least one of
One or more nucleotide insertions in the genome,
one or more nucleotides in the genome are deleted,
one or more nucleotide insertions and deletions in the genome, and
one or more nucleotide substitutions in the genome.
Technical Field
The present invention relates to targeted genome editing in eukaryotic cells or organisms. More specifically, the present invention relates to a composition comprising a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or Cas protein for cleaving the target DNA in a eukaryotic cell or organism and uses thereof.
Background
CRISPR (clustered regularly interspaced short palindromic repeats) is a locus containing multiple short direct repeats that is found in the genome of about 40% sequenced bacteria and 90% sequenced archaea. CRISPRs function as the prokaryotic immune system, conferring resistance to foreign genetic elements such as plasmids and phages. The CRISPR system provides an acquired form of immunity. Short segments of foreign DNA (called spacers) are integrated in the genome between CRISPR repeats as memory of past exposures. The CRISPR spacer is then used to recognize and silence foreign genetic elements in a manner similar to RNAi in eukaryotes.
Cas9, an important protein component of type II CRISPR/Cas systems, when complexed with two RNAs, called CRISPR RNA (crRNA) and trans-activated crRNA (tracrrna), forms an active endonuclease that cleaves an exogenous genetic element in an invading phage or plasmid to protect the host cell. The crRNA is transcribed from a CRISPR element in the host genome, wherein the CRISPR element was previously captured from an exogenous invader. Recently, Jinek et al (1) demonstrated that a single-stranded chimeric RNA generated by fusing essential parts of crRNA and tracrRNA can replace both RNAs in Cas9/RNA complex to form a functional endonuclease.
The CRISPR/Cas system offers advantages over zinc fingers and transcription activator-like effector DNA binding proteins-because site specificity in nucleotide-binding CRISPR-Cas proteins is regulated by RNA molecules rather than DNA-binding proteins (which is more challenging to design and synthesize).
However, to date, no genome editing method has been developed using RNA Guide Endonucleases (RGENs) based on CRISPR/Cas system.
Meanwhile, Restriction Fragment Length Polymorphism (RFLP), one of the oldest, most convenient, and least expensive genotyping methods, is still widely used in molecular biology and genetics, but is often limited by the lack of appropriate restriction enzyme recognition sites.
Mutations induced by engineered nucleases can be detected by a variety of methods including mismatch-sensitive T7 endonuclease I (T7E1) or Surveyor nuclease assay, RFLP, capillary electrophoresis of fluorescent PCR products, dideoxy sequencing and deep sequencing. The T7E1 and Surveyor assays are widely used, but are cumbersome. Furthermore, these enzymes tend to underestimate mutation frequency because the mutated sequences may form homoduplexes with each other, and thus homozygous biallelic mutant clones cannot be distinguished from wild-type cells. RFLP does not have these limitations and is therefore the preferred method. Indeed, RFLP is one of the earliest methods for detecting mutations in cells and animals mediated by engineered nucleases. Unfortunately, however, RFLP is limited by the availability of appropriate restriction sites. There may be no restriction sites at the target site of interest.
Disclosure of Invention
Technical problem
To date, no methods have been developed for genome editing and genotyping using RNA Guide Endonucleases (RGENs) based on CRISPR/Cas systems.
Under such circumstances, the present inventors have made extensive efforts to develop a genome editing method based on CRISPR/Cas system, and finally established a programmable RNA guide endonuclease that can cleave DNA in a targeted manner in eukaryotic cells and organisms.
In addition, the present inventors have made extensive efforts to develop a novel method of using RNA-guided endonuclease (RGEN) in RFLP analysis. Which utilizes RGEN to genotype frequent mutations found in cancer and induced in cells and organisms by engineered nucleases, including RGEN itself, and thus completed the present invention.
Technical scheme
It is an object of the present invention to provide a composition for cleaving a target DNA in a eukaryotic cell or organism, comprising a guide RNA or a DNA encoding a guide RNA specific for the target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a composition for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a kit for cleaving a target DNA in a eukaryotic cell or organism comprising a guide RNA or a DNA encoding a guide RNA specific for the target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a kit for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is a further object of the invention to provide a method of preparing a eukaryotic cell or organism containing a Cas protein and a guide RNA, said method comprising the step of co-transfecting or sequentially transfecting the eukaryotic cell or organism with a Cas protein encoding nucleic acid or Cas protein and a guide RNA or DNA encoding a guide RNA.
It is another object of the invention to provide a eukaryotic cell or organism containing a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the present invention to provide a method for cleaving a target DNA in a eukaryotic cell or organism, said method comprising the steps of: eukaryotic cells or organisms containing a target DNA are transfected with a composition comprising a guide RNA or DNA encoding a guide RNA specific for the target DNA and a Cas protein encoding nucleic acid or Cas protein.
It is another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism, said method comprising the steps of: treating a eukaryotic cell or organism with a composition, wherein the composition contains a guide RNA or DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or Cas protein.
It is a further object of the invention to provide an embryo, a genome modified animal or a genome modified plant comprising a genome edited by a composition comprising a guide RNA specific for a target DNA or a DNA encoding a guide RNA and a Cas protein encoding nucleic acid or Cas protein.
It is another object of the present invention to provide a method for preparing a genomically modified animal, the method comprising the steps of: introducing into an animal embryo a composition comprising a guide RNA or DNA encoding a guide RNA specific for a target DNA and a Cas protein-encoding nucleic acid or Cas protein; and transferring the embryo into the oviduct of a pseudopregnant surrogate mother to produce a genomically modified animal.
It is another object of the invention to provide a composition for genotyping mutations or variations in an isolated biological sample, the composition comprising a guide RNA specific for a target DNA sequence and a Cas protein.
It is another object of the invention to provide a method of genotyping a mutation or naturally occurring mutation or variation in a cell induced by an engineered nuclease using an RNA Guide Endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific to a target DNA and a Cas protein.
It is another object of the invention to provide a kit for genotyping mutations or naturally occurring mutations or variations induced by an engineered nuclease in a cell, the kit comprising an RNA-guided endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for a target DNA and a Cas protein.
It is an object of the present invention to provide a composition for cleaving a target DNA in a eukaryotic cell or organism, said composition comprising a guide RNA or a DNA encoding a guide RNA specific for the target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a composition for inducing targeted mutagenesis in a eukaryotic cell or organism, said composition comprising a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a kit for cleaving a target DNA in a eukaryotic cell or organism, said kit comprising a guide RNA or a DNA encoding a guide RNA specific for the target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a kit for inducing targeted mutagenesis in a eukaryotic cell or organism, said kit comprising a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a method for preparing a eukaryotic cell or organism containing a Cas protein and a guide RNA, said method comprising the step of co-transfecting or sequentially transfecting a eukaryotic cell or organism with a Cas protein encoding nucleic acid or Cas protein and a guide RNA or DNA encoding a guide RNA.
It is another object of the invention to provide a eukaryotic cell or organism containing a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a method for cleaving a target DNA in a eukaryotic cell or organism, the method comprising the step of transfecting the eukaryotic cell or organism containing the target DNA with a composition comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide a method of inducing targeted mutagenesis in a eukaryotic cell or organism, the method comprising the step of treating the eukaryotic cell or organism with a composition comprising a guide RNA or a DNA encoding a guide RNA specific for a target DNA and a Cas protein encoding nucleic acid or a Cas protein.
It is another object of the invention to provide an embryo, a genome modified animal, or a genome modified plant comprising a genome edited by a composition comprising a guide RNA specific for a target DNA or DNA encoding a guide RNA and a Cas protein encoding nucleic acid or Cas protein.
It is another object of the present invention to provide a method for preparing a genomically modified animal, the method comprising the steps of: introducing into an animal embryo a composition comprising a guide RNA or DNA encoding a guide RNA specific for a target DNA and a Cas protein-encoding nucleic acid or Cas protein; and transferring the embryo into the oviduct of a pseudopregnant surrogate mother to produce a genomically modified animal.
It is another object of the invention to provide a composition of genotyping mutations or variations in an isolated biological sample, the composition comprising a guide RNA specific for a target DNA sequence and a Cas protein.
It is another object of the invention to provide a composition for genotyping a nucleic acid sequence of a pathogenic microorganism in an isolated biological sample, the composition comprising a guide RNA specific for a target DNA sequence and a Cas protein.
It is another object of the invention to provide a kit for genotyping mutations or variations in an isolated biological sample, the kit comprising a composition, in particular an RNA-guided endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for the target DNA and a Cas protein.
It is another object of the invention to provide a method of genotyping a mutation or variation in an isolated biological sample using a composition, in particular the composition comprising an RNA Guide Endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for the target DNA and a Cas protein.
Advantageous effects
The inventive compositions for cleaving a target DNA or inducing targeted mutagenesis in a eukaryotic cell or organism comprising a guide RNA specific for the target DNA and a Cas protein encoding nucleic acid or Cas protein, the inventive kits comprising said compositions, and the inventive methods of inducing targeted mutagenesis provide new and convenient genome editing tools. In addition, because custom-made RGENs can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed by RGEN-mediated RFLP, and thus, the compositions and methods of the present invention can be used to detect and cleave naturally occurring variations and mutations.
Brief Description of Drawings
Figure 1 shows Cas 9-catalyzed in vitro plasmid DNA cleavage. (a) Schematic representation of target DNA and chimeric RNA sequences. Red triangles indicate cleavage sites. The PAM sequence recognized by Cas9 is shown in bold. Sequences derived from crRNA and tracrRNA in the guide RNA are shown in boxes and underlined, respectively. (b) Cas9 cleaves plasmid DNA in vitro. The whole circular plasmid or ApaLI digested plasmid was incubated with Cas9 and guide RNA.
Figure 2 shows Cas9 induced mutagenesis at an episomal (episomal) target site. (a) Schematic representation of cell-based assays using RFP-GFP reporter. Because the GFP sequence is fused out of frame to the RFP sequence, GFP is not expressed from the reporter. The RFP-GFP fusion protein is expressed only when the target site between the two sequences is cleaved by a site-specific nuclease. (b) Flow cytometry of Cas9 transfected cells. The percentage of cells expressing the RFP-GFP fusion protein is shown.
FIG. 3 shows RGEN-driven mutations at endogenous chromosomal sites. (a) The CCR5 locus. (b) The C4BPB locus. (top) RGEN driven mutations were detected using the T7E1 assay. The arrow indicates the expected position of the DNA band cut by T7E 1. The mutation frequency (Indel (%)) was calculated by measuring the band intensity. DNA sequences of (bottom) CCR5 and C4BPB Wild Type (WT) and mutant clones. The region of the target sequence complementary to the guide RNA is indicated in frame (in boc). The PAM sequence is shown in bold. Triangles indicate cleavage sites. Bases corresponding to micro-homology (microhomologies) are underlined. The right hand column shows the number of inserted or deleted bases.
FIG. 4 shows that no RGEN-driven off-target (off-target) mutations could be detected. (a) Target (On-target) sequences and potential off-target sequences. The human genome was searched on silicon chips for potential off-target sites. Four sites were identified, each of which carried a3 base mismatch to the CCR5 target site. Mismatched bases are underlined. (b) The T7E1 assay was used to investigate whether these sites are mutated in cells transfected with the Cas9/RNA complex. No mutations were detected at these sites. N/A (not applicable), intergenic site. (c) Cas9 did not induce off-target related chromosomal deletions. RGEN and ZFN specific for CCR5 are expressed in human cells. The induction of the 15-kb chromosomal deletion in these cells was examined using PCR.
FIG. 5 shows RGEN-induced Foxn1 gene targeting in mice. (a) A schematic of sgrnas specific for exon 2 of the mouse Foxn1 gene is depicted. PAM in exon 2 is shown in red and the sequence in the sgRNA that is complementary to exon 2 is underlined. Triangles indicate cleavage sites. (b) representative T7E1 assay, demonstrating gene targeting efficiency of Cas9mRNA + Foxn1 specific sgRNA in mouse embryos delivered to one cell stage by intracytoplasmic injection. Numbers indicate independent founder mice produced by the highest dose. The arrow indicates the strip cut by T7E 1. (c) The DNA sequences of the mutant alleles observed in the three Foxn1 mutant founder mice identified in b. The occurrence numbers are shown in parentheses. (d) PCR genotyping of F1 progeny resulting from Foxn1 founder mouse #108 and wild type FVB/NTac crosses. Note that the mutant allele present in Foxn1 founder mouse #108 was isolated in the offspring.
Figure 6 shows Foxn1 gene targeting in mouse embryos by intracytoplasmic injection of Cas9mRNA and Foxn 1-sgRNA. (a) Results of a representative T7E1 assay, which monitored mutation rate after injection of the highest dose. The arrow indicates the strip cut by T7E 1. (b) And (6) summarizing the detection result of T7E 1. Mutant scores in vitro cultured embryos obtained after intracytoplasmic injection of the indicated dose of RGEN are shown. (c) DNA sequence of Foxn1 mutant alleles identified from T7E1 positive mutant embryo subsets. The target sequence for the wild-type allele is shown in frame.
Figure 7 shows Foxn1 gene targeting in mouse embryos using recombinant Cas9 protein, Foxn1-sgRNA complex. (a) And (b) is representative T7E1 assay results and summaries thereof. The embryos are cultured in vitro after prokaryotic (a) or intracytoplasmic injection (b). Red numbers indicate T7E1 positive mutation founder mice. (c) DNA sequence of the Foxn1 mutant allele identified from embryos cultured in vitro by prokaryotic injection of recombinant Cas9 protein at the highest dose: foxn1-sgRNA complex. The target sequence for the wild-type allele is shown in frame.
Fig. 8 shows germline propagation of the mutant allele found in Foxn1 mutant builder # 12. (a) fPCR analysis. (b) PCR genotyping wild-type FVB/NTac, founder mice and their F1 progeny.
Figure 9 shows the genotypes of embryos generated by crossing Prkdc mutation founder mice. Prkcc mutation founder mice male and female 25 and 15 cross and E13.5 embryos isolated. (a) fPCR analysis of wild type, founder mouse male 25, founder mouse female 15. It is noted that due to the technical limitations of the fPCR analysis, these results show minor differences from the exact sequence of the mutant allele; for example, from sequence analysis, Δ 269/Δ 61/WT and Δ 5+1/+7/+12/WT were identified in founder mice, male and female, 25 and 15, respectively. (b) The genotype of the embryo produced.
Fig. 10 shows targeted mutations induced by Cas9 protein/sgRNA complex.
Figure 11 shows mutations induced in arabidopsis protoplasts by recombinant Cas9 protein.
Figure 12 shows the sequence of mutations induced by the recombinant Cas9 protein in the arabidopsis BRI1 gene.
Fig. 13 shows a T7E1 assay, which shows disruption of the endogenous CCR5 gene by treatment with Cas9-mal-9R4L and sgRNA/C9R4LC complex in 293 cells.
FIG. 14(a, b) shows the mutation frequencies of RGENs at the target and off-target sites reported by Fu et al (2013). The T7E1 assay analyzed genomic DNA from K562 cells sequentially transfected with 20. mu.g Cas9 encoding plasmid and 60. mu.g and 120. mu.g in vitro transcribed GX19 crRNA and tracrRNA (1X 10. mu.g and 120. mu.g, respectively)6Individual cells) (R), or co-transfected with 1. mu.g Cas9 encoding plasmid and 1. mu.g GX19sgRNA expression plasmid (2X 10)5Individual cells) (D).
FIG. 15(a, b) shows a comparison of guide RNA structures. The mutation frequencies of RGENs reported by Fu et al (2013) on target and off-target sites were measured using the T7E1 assay. K562 cells were co-transfected with Cas9 encoding plasmids and plasmids encoding GX19 sgRNA or GGX20 sgRNA. Off-target sites (OT1-3, etc.) are labeled as in Fu et al (2013).
Figure 16 shows Cas9 nickase DNA cleavage in vitro. (a) Schematic representation of Cas9 nuclease and paired Cas9 nickase. The PAM sequence and cleavage site are shown in boxes. (b) A target site in the human AAVS1 locus. The position of each target site is represented by a triangle. (c) Schematic representation of the DNA cleavage reaction. FAM dyes (shown in box) are attached to both 5' ends of the DNA substrate. (d) DSB and SSB were analyzed using fluorescence capillary electrophoresis. The fluorescently labeled DNA substrate is incubated with Cas9 nuclease or nickase prior to electrophoresis.
Figure 17 shows a comparison of Cas9 nuclease and nickase behavior. (a) Target mutation frequencies associated with Cas9 nuclease (WT), nickase (D10A), and paired nickase. Showing the paired nickase enzyme that produces either 5 'or 3' overhangs. (b) Analysis of off-target effects of Cas9 nuclease and paired nickase. A total of 7 potential off-target sites for three sgrnas were analyzed.
Figure 18 shows paired Cas9 nickases tested at other endogenous human loci. (a, c) sgRNA target site at human CCR5 and BRCA2 loci. The PAM sequence is shown in red. (b, d) detecting genome editing activity at each target site by T7E1 assay. Repair of two gaps that produce 5 'overhangs results in much more frequent indel formation than repair of two gaps that produce 3' overhangs.
Figure 19 shows paired Cas9 nickase mediated homologous recombination. (a) And (3) detecting the strategy of homologous recombination. The donor DNA includes an XbaI restriction site between the two homologous arms, while the endogenous target site lacks this restriction site. PCR assays are used to detect sequences that have undergone homologous recombination. To prevent amplification of contaminating donor DNA, primers specific for genomic DNA are used. (b) efficiency of homologous recombination. Only the amplicon of the region where homologous recombination has occurred can be digested with XbaI; the strength of the cut strip was used to measure the efficiency of the method.
Figure 20 shows DNA splicing induced by paired Cas9 nickase. (a) Target sites for paired nickases in the human AAVS1 locus. The distance between the AS2 site and each of the other sites is shown. Arrows indicate PCR primers. (b) Genomic deletions were detected by PCR. Asterisks indicate deletion-specific PCR products. (c) DNA sequence of deletion-specific PCR product obtained using AS2 and L1 sgRNA. The target site PAM sequence is shown in frame and the sgRNA-matching sequence is in capital letters. The complete sgRNA-matching sequence is underlined. (d) Schematic model of paired Cas9 nickase-mediated chromosomal deletion. The newly synthesized DNA strand is shown in box.
Figure 21 shows that paired Cas9 nickase does not induce translocation. (a) Schematic representation of chromosomal translocations between target and off-target sites. (b) PCR amplification to detect chromosomal translocations. (c) Translocation induced by Cas9 nuclease, but not by nickase pairs.
FIG. 22 shows a conceptual diagram of T7E1 and RFLP assays. (a) Comparison of cleavage reactions was determined in four possible cases after engineered nuclease treatment in diploid cells: (A) wild type, (B) single allele mutation, (C) different double allele mutation (heterozygous), and (D) the same double allele mutation (homozygous). Black lines represent PCR products derived from each allele; short dashed boxes and dotted boxes indicate insertion/deletion mutations generated by NHEJ. (b) Expected results of T7E1 and RGEN digests resolved by electrophoresis.
FIG. 23 shows an in vitro cleavage assay of linearized plasmids containing the C4BPB target site (with indels). DNA sequence of each plasmid substrate (upper panel). The PAM sequence is underlined. The inserted bases are shown in frame. The arrow (lower panel) indicates the expected position of the DNA band cleaved with wild-type specific RGEN after electrophoresis.
FIG. 24 shows genotyping of engineered nuclease-induced mutations in cells by RGEN-mediated RFLP. (a) Genotype of C4BPB mutant K562 cell clone. (b) The mismatch sensitive T7E1 assay was compared to RGEN-mediated RFLP analysis. Black arrows indicate cleavage products treated by T7E1 enzyme or RGEN.
FIG. 25 shows mutations induced by genotyping RGEN by RGEN-RFLP techniques. (a) C4 BPB-disrupted clones were analyzed using the RGEN-RFLP and T7E1 assays. The arrow indicates the expected position of the DNA band cleaved by RGEN or T7E 1. (b) Quantitative comparison of RGEN-RFLP analysis with the T7E1 assay. Genomic DNA samples from wild-type and C4 BPB-disrupted K562 cells were mixed in different ratios and subjected to PCR amplification. (c) Genotyping of RGEN-induced mutations in HLA-B genes in HeLa cells using RFLP and T7E1 assays.
FIG. 26 shows genotyping of engineered nuclease-induced mutations in organisms by RGEN-mediated RFLP. (a) The Pibf1 mutant establishes the genotype of the mice. (b) The mismatch sensitive T7E1 assay was compared to RGEN-mediated RFLP analysis. Black arrows indicate T7E1 enzyme or RGEN treated cleavage products.
Figure 27 shows RGEN-mediated genotyping of ZFN-induced mutations. ZFN target sites are shown in box. The black arrows indicate the DNA bands cleaved by T7E 1.
FIG. 28 shows polymorphic sites in a human HLA-B gene region. The sequence surrounding the RGEN target site is the sequence of the PCR amplicon from HeLa cells. The polymorphism locations are shown in boxes. The RGEN target sites and PAM sequences are shown in dashed and bold boxes, respectively. The primer sequences are underlined.
FIG. 29 shows genotyping of oncogenic mutations by RGEN-RFLP analysis. (a) Frequent mutations in the human CTNNB1 gene (c.133-135 deletion of TCT) were detected in HCT116 cells by RGEN. HeLa cells were used as negative control. (b) The mutation was replaced by genotyping KRAS in a549 cancer cell line with RGEN containing mismatch guide RNA (c.34g > a). Mismatched nucleotides are shown in boxes. HeLa cells were used as negative control. The arrows indicate the DNA bands cleaved by RGEN. DNA sequences confirmed by Sanger sequencing are shown.
FIG. 30 shows genotyping of the CCR5 delta32 allele in HEK293T cells by RGEN-RFLP analysis. (a) RGEN-RFLP assay of cell lines. K562, SKBR3 and HeLa cells were used as wild type controls. The arrows indicate the RGEN-cleaved DNA bands. (b) DNA sequences of the wild type and delta32 CCR5 allele. The targets and off-target sites of RGENs used in RFLP analysis are underlined. Single nucleotide mismatches between the two sites are shown in frame. The PAM sequence is underlined. (c) Plasmids carrying the WT or del32 CCR5 allele were cleaved in vitro using wild-type specific RGENs. (d) The presence of a off-target site for RGEN specific for CCR5-delta32 at the CCR5 locus was confirmed. In vitro cleavage assays were performed on plasmids bearing either target or off-target sequences using various amounts of del 32-specific RGENs.
FIG. 31 shows genotyping of KRAS point mutations (c.34G > A). (a) RGEN-RFLP analysis of KRAS mutations (c.34G > A) in cancer cell lines. PCR products from HeLa cells (used as wild-type control) or a549 cells (homozygous for point mutation) were digested with RGENs with perfectly matched crRNA specific for either wild-type or mutant sequences. KRAS genotype was confirmed by Sanger sequencing in these cells. (b) Plasmid (which carries wild-type or mutated KRAS sequence) was digested with RGEN with perfectly matched crRNA or attenuated single base mismatched crRNA. The attenuated crrnas selected for genotyping are indicated in boxes above the gel.
FIG. 32 shows PIK3CA point mutation (c.3140A > G) genotyping. (a) RGEN-RFLP analysis of PIK3CA mutations (c.3140A > G) in cancer cell lines. PCR products from HeLa cells (used as wild-type control) or HCT116 cells (heterozygous point mutation) were digested with RGENs with perfectly matched crRNA specific for either the wild-type sequence or the mutated sequence. PIK3CA genotype was confirmed by Sanger sequencing in these cells. (b) Plasmids carrying the wild-type or mutant PIK3CA sequences were digested with RGENs with perfectly matched crRNA or attenuated single base mismatched crRNA. The attenuated crrnas selected for genotyping are indicated in boxes above the gel.
FIG. 33 shows genotyping of frequency point mutations in cancer cell lines. RGEN-RFLP measures recurrent cancer point mutations in (a) IDH (c.394c > T), (b) PIK3CA (c.3140A > G), (c) NRAS (c.181C > A), and (d) the BRAF gene (c.1799T > A). The genotype of each cell line as confirmed by Sanger sequencing is shown. Mismatched nucleotides are shown in boxes. The black arrows indicate the DNA bands cleaved by RGEN.
PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION
According to one aspect of the invention, the invention provides a composition for cleaving a target DNA in a eukaryotic cell or organism, comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA, and a Cas protein encoding nucleic acid or a Cas protein. In addition, the invention provides the use of such a composition comprising a guide RNA specific for a target DNA or a DNA encoding such a guide RNA, and a Cas protein encoding nucleic acid or a Cas protein for cleaving the target DNA in a eukaryotic cell or organism.
In the present invention, the composition is also referred to as an RNA-guided endonuclease (RGEN) composition.
ZFNs and TALENs enable targeted mutagenesis in mammalian cells, model organisms, plants and livestock, but the mutation frequencies obtained with each nuclease differ greatly from each other. Furthermore, some ZFNs and TALENs do not show any genome editing activity. DNA methylation may limit binding of these engineered nucleases to the target site. Furthermore, generating custom nucleases is technically challenging and time consuming.
The present inventors have developed new RNA-guided endonuclease compositions based on Cas proteins to overcome the disadvantages of ZFNs and TALENs.
Prior to the present invention, the endonuclease activity of Cas proteins was known. However, due to the complexity of the eukaryotic genome, it is not known whether the endonuclease activity of Cas proteins will play a role in eukaryotic cells. Furthermore, to date, no composition comprising a Cas protein or a Cas protein-encoding nucleic acid and a guide RNA specific for a target DNA has been developed for cleaving a target DNA in a eukaryotic cell or organism.
The present RGEN compositions based on Cas proteins can be more easily customized than ZFNs and TALENs because: to generate a new genome editing nuclease, only the synthetic guide RNA component may be replaced. No subcloning step is involved to generate a custom RNA guide endonuclease. Furthermore, the relatively small size of the Cas gene (e.g., 4.2kbp for Cas9) compared to a pair of TALEN genes (-6 kbp) provides advantages for this RNA-guided endonuclease composition in some applications, such as virus-mediated gene delivery. In addition, the RNA has no off-target effect on the guide endonuclease, and thus does not cause unwanted mutations, deletions, inversions, and duplications. These properties make the RNA-guided endonuclease compositions of the present invention an extensible, versatile and convenient tool for genome engineering in eukaryotic cells and organisms. In addition, RGENs can be designed to target any DNA sequence, and virtually any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed by RGEN-mediated RFLP. The specificity of RGEN is determined by the RNA component that hybridizes to a target DNA sequence of no more than 20 base pairs (bp) in length and the Cas9 protein, which Cas9 protein recognizes a protospacer-adjacencies motif (PAM). RGENs can be easily reprogrammed by replacing RNA components. Accordingly, RGEN provides a platform for simple robust RFLP analysis for a variety of sequence variations.
The target DNA may be endogenous DNA or artificial DNA, preferably endogenous DNA.
As used herein, the term "Cas protein" refers to an essential protein component in the CRISPR/Cas system, which forms an active endonuclease or nickase when complexed with 2 RNAs referred to as CRISPR RNA (crRNA) and trans-activating crRNA (tracrrna).
Information on Cas genes and proteins is available from GenBank of the National Center for Biotechnology Information (NCBI), without limitation.
CRISPR-associated (Cas) genes encoding Cas proteins are typically associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described. Among these protein families, Cas1 appears to be spread throughout the various CRISPR/Cas systems. There are three types of CRISPR-Cas systems. Among them, the type II CRISPR/Cas system involving Cas9 protein and crRNA and tracrRNA is representative and well known. Specific combinations of cas genes and repeat structures were used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, tnepap, Hmari, aperrn, and Mtube).
The Cas protein may be linked to a protein transduction domain. The protein transduction domain may be polyarginine or a TAT protein derived from HIV, but is not limited thereto.
The inventive compositions can comprise a Cas component in the form of a protein or a nucleic acid encoding a Cas protein.
In the present invention, the Cas protein may be any Cas protein as long as it has endonuclease or nickase activity when it is complexed with the guide RNA.
Preferably, the Cas protein is a Cas9 protein or a variant thereof.
A variant of Cas9 protein may be a mutant form of Cas9 in which the catalytic aspartate residue is changed to any other amino acid. Preferably, the other amino acid may be alanine, but is not limited thereto.
Furthermore, the Cas protein may be a protein isolated from an organism such as Streptococcus species (Streptococcus sp.), preferably Streptococcus pyogenes (Streptococcus pyogens), or a recombinant protein, but is not limited thereto.
Cas proteins derived from streptococcus pyogenes recognize NGG trinucleotides. The Cas protein may include SEQ id no: 109, but is not limited thereto.
The term "recombinant," when used with reference, for example, to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant Cas protein can be produced by reconstructing a Cas protein coding sequence using a human codon table.
For the purposes of the present invention, the Cas protein-encoding nucleic acid may be in the form of a vector, such as a plasmid comprising the Cas-encoding sequence under a promoter, such as CMV or CAG. When the Cas protein is Cas9, the Cas9 coding sequence may be derived from streptococcus, preferably from streptococcus pyogenes. For example, the Cas9 encoding nucleic acid may comprise SEQ ID NO: 1. Furthermore, the Cas9 encoding nucleic acid may comprise a sequence identical to SEQ ID NO: 1, preferably a nucleotide sequence having at least 50% homology to the sequence of SEQ ID NO: 1 has at least 60, 70, 80, 90, 95, 97, 98, or 99% homology, but is not limited thereto. Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID nos.108, 110, 106 or 107.
As used herein, the term "guide RNA" refers to an RNA specific for a target DNA that can form a complex with a Cas protein and bring the Cas protein to the target DNA.
In the present invention, the guide RNA may be composed of two RNAs, i.e., CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or the guide RNA may be a single-stranded RNA (sgrna) produced by fusing essential parts of crRNA and tracrRNA.
The guide RNA may be a double RNA (dualrna) comprising crRNA and tracrRNA.
Any guide RNA may be used in the present invention if it contains the necessary parts of crRNA and tracrRNA and the part complementary to the target.
The crRNA may hybridize to the target DNA.
RGENs can consist of a Cas protein and a dualRNA (invariant tracrRNA and target-specific crRNA), or a Cas protein and an sgRNA (fusion of invariant tracrRNA and an essential part of target-specific crRNA), and can be easily reprogrammed by replacing the crRNA.
The guide RNA may further comprise one or more additional nucleotides at the 5' end of the crRNA of the single stranded guide RNA or the dualRNA.
Preferably, the guide RNA may further comprise 2 additional guanine nucleotides at the 5' end of the crRNA of the single stranded guide RNA or the dualRNA.
The guide RNA may be transferred into the cell or organism in the form of RNA or DNA encoding the guide RNA. The guide RNA may be in the form of isolated RNA, RNA incorporated into a viral vector, or encoded in a vector. Preferably, the vector may be a viral vector, a plasmid vector, or an agrobacterium vector, but is not limited thereto.
The DNA encoding the guide RNA may be a vector comprising a sequence encoding the guide RNA. For example, a guide RNA can be transfected into a cell or organism by transfecting the cell or organism with an isolated guide RNA or plasmid DNA comprising a sequence encoding a guide RNA and a promoter.
Alternatively, viral-mediated gene delivery can be used to transfer guide RNAs to cells or organisms.
When the guide RNA is transfected into a cell or organism in the form of an isolated RNA, the guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA is preferably transferred to the cell in the form of an isolated RNA, rather than in the form of a plasmid containing the coding sequence of the guide RNA. As used herein, the term "isolated RNA" is used interchangeably with "naked RNA". This saves cost and time because no cloning step is required. However, transfection of guide RNA using plasmid DNA or virus-mediated gene delivery is not excluded.
Due to the specificity of the guide RNA for the target and the endonuclease or nickase activity of the Cas protein, the RGEN composition of the invention comprising the Cas protein or Cas protein encoding nucleic acid and the guide RNA can specifically cleave the target DNA.
As used herein, the term "cleavage" refers to the cleavage of the covalent backbone of a nucleotide molecule.
In the present invention, guide RNAs can be prepared to be specific for any target to be cleaved. Thus, the RGEN compositions of the invention can cleave any target DNA by manipulating or genotyping a target-specific portion of the guide RNA.
The guide RNA and Cas protein may act as pairs (pair). As used herein, the term "paired Cas nickase" may refer to a guide RNA and a Cas protein that act as pairs. The pair (pair) comprises two guide RNAs. The guide RNA and Cas protein can act as pairs, inducing two gaps on different DNA strands. The two gaps can be separated by at least 100 bps, but are not limited thereto.
In the examples, the inventors demonstrated that paired Cas nickases allow targeted mutagenesis and large deletions of chromosomal fragments up to 1-kbp to be achieved in human cells. Importantly, paired nickases do not induce indels at off-target sites, whereas their corresponding nucleases induce mutations at off-target sites. In addition, unlike nucleases, paired nickases do not promote unwanted translocations associated with off-target DNA cleavage. In principle, paired nickases double the specificity of Cas 9-mediated mutagenesis, and can expand the use of RNA guides in applications requiring precise genome editing (such as gene and cell therapies).
In the present invention, the compositions are useful for in vitro genotyping the genome of a eukaryotic cell or organism.
In a specific embodiment, the guide RNA may comprise the nucleotide sequence of SEQ ID No.1, wherein the portion of nucleotide positions 3 to 22 is a target-specific portion, and thus the sequence of this portion may vary depending on the target.
As used herein, without limitation, eukaryotic cells or organisms may be yeast, fungal, protozoan, plant, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, e.g., cultured cells (in vitro), transplanted cells and primary cell cultures (in vitro and ex vivo), and in vivo cells, as well as mammalian cells commonly used in the art, including human cells.
In a specific embodiment, it was found that Cas9 protein/single stranded guide RNA can generate site-specific DNA double strand breaks in vitro and in mammalian cells, whose spontaneous repair induces targeted genomic mutations with high frequency.
Furthermore, it was found that knockout mice can be induced by injecting Cas9 protein/guide RNA complex or Cas9 mRNA/guide RNA into one-cell stage embryos and germline transmissible mutations can be generated by Cas 9/guide RNA system.
It is advantageous to use a Cas protein instead of a nucleic acid encoding a Cas protein to induce targeted mutagenesis, since no foreign DNA is introduced into the organism. Thus, compositions comprising a Cas protein and a guide RNA may be used to develop therapeutics or value-added crops, livestock, poultry, fish, pets, and the like.
According to another aspect of the invention, the invention provides a composition for inducing site-directed mutagenesis in a eukaryotic cell or organism comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein. In addition, the invention provides the use of a composition comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein for inducing targeted mutagenesis in a eukaryotic cell or organism.
The guide RNA, Cas protein-encoding nucleic acid, or Cas protein are described above.
According to another aspect of the invention, the invention provides a kit for cleaving a target DNA or inducing targeted mutagenesis in a eukaryotic cell or organism, comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or Cas protein.
Guide RNAs, Cas protein-encoding nucleic acids or Cas proteins are described above.
The kit may comprise the guide RNA and the Cas protein-encoding nucleic acid or Cas protein as separate components or as one composition.
The kit of the invention may comprise some other components necessary for transferring the guide RNA and Cas component to the cell or organism. For example, the kit may include an injection buffer, such as DEPC-treated injection buffer, and substances necessary for analyzing a mutation of the target DNA, but is not limited thereto.
According to another aspect, the invention provides a method of preparing a eukaryotic cell or organism comprising a Cas protein and a guide RNA, said method comprising the step of co-transfecting or sequentially transfecting the eukaryotic cell or organism with a Cas protein encoding nucleic acid or a Cas protein and a guide RNA or DNA encoding said guide RNA.
Guide RNAs, Cas protein-encoding nucleic acids or Cas proteins are described above.
In the present invention, the Cas protein-encoding nucleic acid or Cas protein and the guide RNA or DNA encoding the guide RNA may be transferred into cells by various methods known in the art, such as microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transduction, virus-mediated gene delivery, and PEG-mediated protoplast transfection, and the like, but not limited thereto. In addition, the Cas protein-encoding nucleic acid or Cas protein and guide RNA can be transferred to an organism by various methods of administering the gene or protein known in the art (e.g., injection). The Cas protein-encoding nucleic acid or Cas protein may be transferred into the cell in the form of a complex with a guide RNA, or separately. Cas proteins fused to protein transduction domains (such as Tat) can also be efficiently delivered into cells.
Preferably, the eukaryotic cell or organism is co-transfected or sequentially transfected with a Cas9 protein and a guide RNA.
Sequential transfection may be performed as follows: a first transfection with Cas protein-encoding nucleic acid followed by a second transfection with naked guide RNA. Preferably, the second transfection is after 3, 6, 12, 18, 24 hours, but is not limited thereto.
According to another aspect, the invention provides a eukaryotic cell or organism comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein.
Eukaryotic cells or organisms can be prepared by transferring a composition comprising a guide RNA specific for a target DNA or DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein into the cell or organism.
Eukaryotic cells may be yeast, fungal, protozoan, higher plant, insect, or amphibian cells or mammalian cells, such as CHO, HeLa, HEK293, and COS-1, e.g., cultured cells (in vitro), transplanted cells and primary cell cultures (in vitro and ex vivo), and in vivo cells, as well as mammalian cells commonly used in the art, including human cells, and the like, without limitation. Further the organism may be a yeast, fungus, protozoa, plant, higher plant, insect, amphibian, or mammal.
According to another aspect of the invention, the invention provides a method for cleaving a target DNA or inducing targeted mutagenesis in a eukaryotic cell or organism, the method comprising the step of treating the cell or organism containing the target DNA with a composition comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein.
The step of treating the cell or organism with a composition comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein can be performed by transferring the composition of the invention into the cell or organism.
As described above, such transfer may be performed by microinjection, transfection, electroporation, or the like.
According to another aspect of the invention, the invention provides an embryo comprising a genome edited by an RGEN composition of the invention comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein.
Any embryo can be used in the present invention, for which the embryo can be a mouse embryo. Embryos can be produced by: superovulated female mice can be mated with male mice by injecting PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin) into female mice for 4-7 weeks, and fertilized embryos can be collected from the oviducts.
The RGEN compositions of the invention introduced into the embryo can cleave the target DNA complementary to the guide RNA by the action of the Cas protein, causing mutations in the target DNA. Thus, embryos incorporating the RGEN compositions of the invention have edited genomes.
In a particular embodiment, it has been found that the RGEN compositions of the invention can result in mutations in mouse embryos that can be transmitted to offspring.
The method of introducing the RGEN composition into the embryo can be any method known in the art, such as microinjection, stem cell insertion, retroviral insertion, and the like. Preferably, microinjection techniques can be used.
According to another aspect, the invention provides a genome-modified animal obtained by transferring an embryo to the oviduct of the animal, wherein the embryo comprises a genome edited by an RGEN composition of the invention.
In the present invention, the term "genome-modified animal" refers to an animal whose genome has been modified at the embryonic stage with the RGEN composition of the present invention, and the species of the animal is not limited.
Genomically modified animals have mutations caused by targeted mutagenesis based on the RGEN compositions of the invention. The mutation may be any of a deletion, an insertion, a translocation, an inversion. The site of the mutation depends on the guide RNA sequence of the RGEN composition.
A genomically modified animal having a mutation in a gene can be used to determine the function of the gene.
According to another aspect of the invention, the invention provides a method of making a genomically modified animal comprising the step of introducing into an animal embryo a RGEN composition of the invention comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA and a Cas protein encoding nucleic acid or Cas protein; and transferring the embryo to a pseudopregnant surrogate mother oviduct to produce a genome modified animal.
The step of introducing the RGEN compositions of the invention can be accomplished by any method known in the art, such as microinjection, stem cell insertion, retroviral insertion, and the like.
According to another aspect of the invention, plants regenerated from genomically modified protoplasts prepared by the methods for generating eukaryotic cells containing an RGEN composition are provided.
According to another aspect of the invention, the invention provides a composition for genotyping mutations or variations in an isolated biological sample, the composition comprising a guide RNA specific for a target DNA sequence and a Cas protein. In addition, the present invention provides a composition of nucleic acid sequences for genotyping a pathogenic microorganism in an isolated biological sample, the composition comprising a guide RNA specific for a target DNA sequence and a Cas protein.
The guide RNA, Cas protein-encoding nucleic acid, or Cas protein are described above.
The term "genotyping" as used herein refers to "Restriction Fragment Length Polymorphism (RFLP) analysis".
RFLP can be used to 1) detect indels in cells or organisms induced by engineered nucleases, 2) to genotype mutations or variations naturally occurring in cells or organisms, or 3) to genotype the DNA of infected pathogenic microorganisms (including viruses or bacteria, etc.).
Mutations or variations can be induced in cells by engineered nucleases.
The engineered nuclease may be a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an RGEN, but is not limited thereto.
The term "biological sample" as used herein includes, but is not limited to, a sample to be analyzed, such as tissue, cells, whole blood, SEMM, plasma, saliva, sputum, cerebrospinal fluid, or urine.
The mutation or variation may be a naturally occurring mutation or variation.
The mutation or variation is caused by a pathogenic microorganism. That is, a mutation or variation occurs due to infection by a pathogenic microorganism, and when the pathogenic microorganism is detected, the biological sample is identified as infected.
The pathogenic microorganism may be a virus or a bacterium, but is not limited thereto.
Engineered nuclease-induced mutations can be detected by a variety of methods, including mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays, RFLP analysis, fluorescent PCR, DNA melting analysis, and Sanger and deep sequencing (deep sequencing). The T7E1 and Surveyor assays are widely used, but often the mutation frequency is underestimated because these assays can detect heteroduplexes (formed by hybridization of a mutant and wild-type sequence or by hybridization of two different mutant sequences); they are unable to detect homoduplexes formed by the hybridization of two identical mutant sequences. Thus, these assays were unable to distinguish homozygous biallelic mutant clones from wild-type cells, nor heterozygous biallelic mutants from heterozygous single-allelic mutants (FIG. 22). In addition, sequence polymorphisms near the target site of a nuclease can lead to confounding results, as the enzyme can cleave heteroduplexes formed by hybridization of these different wild-type alleles. RFLP analysis does not have these limitations and is therefore the preferred method. Indeed, RFLP analysis was one of the earliest methods for detecting engineered nuclease-mediated mutations. Unfortunately, however, it is limited by the availability of suitable restriction sites.
According to another aspect of the invention, the invention provides a kit for genotyping a mutation or variation in an isolated biological sample, the kit comprising a composition for genotyping a mutation or variation in an isolated biological sample. In addition, the present invention provides a kit for genotyping nucleic acid sequences in a pathogenic microorganism in an isolated biological sample, the kit comprising a guide RNA specific for a target DNA sequence and a Cas protein.
Guide RNAs, Cas protein-encoding nucleic acids or Cas proteins are described above.
According to another aspect of the invention, there is provided a method of genotyping a mutation or variation in an isolated biological sample using the composition for genotyping a mutation or variation in an isolated biological sample. In addition, the present invention provides a method of genotyping nucleic acid sequences in a pathogenic microorganism in an isolated biological sample comprising a guide RNA specific for a target DNA sequence and a Cas protein.
Guide RNAs, Cas protein-encoding nucleic acids or Cas proteins are described above.
Detailed description of the invention
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples.