COMPOSITION FOR BASE EDITING FOR ANIMAL EMBRYO AND BASE EDITING METHOD

Abstract

Provided are a base editing composition comprising deaminase and target-specific nuclease, a base editing method using the base editing composition, and a method for producing a genetically modified animal. The base editing composition has a base editing activity in mammalian embryos.

Claims

1. A base editing composition for a mammalian cell, the composition comprising: a cytidine deaminase or a coding gene therefor; and a target-specific nuclease or a coding gene therefor.

2. The base editing composition of claim 1, wherein the target-specific nuclease comprises an RNA-guided nuclease and a guide RNA.

3. The base editing composition of claim 2, wherein the RNA-guided nuclease is a Cas9 protein or a Cpf1 protein.

4. The base editing composition of claim 2, wherein the RNA-guided nuclease is Cas9 nickase, a catalytically deficient Cas9 protein, or a Cas9 protein that recognizes a PAM sequence different from a wild-type Cas9 protein.

5. The base editing composition of claim 4, wherein the RNA-guided nuclease comprising an amino acid sequence of Streptococcus pyogenes-derived Cas9 protein wherein the following amino acid residues are substituted with amino acid residues different from wild-type amino acid residues: (1) D10, H840, or D10 and H840; (2) at least one selected from the group consisting of D1135, R1335, T1337; or (3) both of (1) and (2) amino acid residues.

6. The base editing composition of claim 2, wherein the guide RNA is a dual RNA or single guide RNA (sgRNA) comprising CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

7. The base editing composition of claim 1, wherein the cytidine deaminase is APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like), AID (activation-induced cytidine deaminase), tadA (tRNA-specific adenosine deaminase), or a combination thereof.

8. The base editing composition of claim 1, wherein the base editing composition for a mammalian cell comprises a cytidine deaminase-encoding mRNA, an RNA-guided nuclease-encoding mRNA, and a guide RNA.

9. The base editing composition of claim 1, wherein the base editing composition for a mammalian cell comprises a ribonucleoprotein in which a cytidine deaminase, an RNA-guided nuclease, and a guide RNA form together a complex.

10. The base editing composition of claim 1, further comprising a uracil DNA a glycosylase inhibitor (UGI) or a coding gene therefor, a nuclear localization sequence (NLS) or a coding gene therefor, or all of them.

11. The base editing composition of claim 1, wherein the mammalian cell is a mammalian embryo.

12. A base editing method for a mammalian cell, the method comprising injecting the base editing composition of claim 1 to the mammalian cell.

13. The base editing method of claim 12, wherein the base editing composition further comprises a uracil DNA a glycosylase inhibitor (UGI) or a coding gene therefor, a nuclear localization sequence (NLS) or a coding gene therefor, or all of them.

14. The base editing method of claim 12, wherein the injecting step is conducted by microinjection or electroporation.

15. The base editing method of claim 12, wherein the mammalian cell is a mammalian embryo.

16. A genetically modified mammalian cell, wherein the base editing composition of claim 1 is injected thereto.

17. The genetically modified mammalian cell of claim 16, wherein base editing composition further comprises a uracil DNA glycosylase inhibitor (UGI) or a coding gene therefor, a nuclear localization sequence (NLS) or a coding gene therefor, or both of them.

18. The genetically modified mammalian cell of claim 16, wherein the mammalian cell is a mammalian embryo.

19. A genetically modified mammal, developed by transplanting the genetically modified mammalian embryo of claim 18 to an oviduct in a mammalian surrogate mother.

20. A method for constructing a genetically modified mammal, the method comprising transplanting the genetically modified mammalian embryo of claim 18 to an oviduct in a mammalian surrogate mother.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIGS. 1a to 1e pertain to dystrophin-deficient mutant mice generated by cytidine deaminase-mediated base editing,

[0130] FIG. 1a shows the nucleotide sequence at the target site in the dystrophin locus (Dmd) (the PAM sequence and the sgRNA target sequence are shown in blue and black, respectively and the nucleotide substituted by cytidine deaminase-mediated base editing is shown in red),

[0131] FIG. 1b shows alignments of nucleotide sequences at the target site in the Dmd gene from newborn pups that developed after base editor 3 (BE3) (rAPOBEC1-nCas9-UGI)-encoding mRNA and sgRNA hybridizable with the target sequence of the Dmd gene of FIG. 1a were microinjected into mouse zygotes (Wt, wild-type; the target sequence is underlined; the PAM sequence and substituted nucleotides are shown in blue and red, respectively; the column on the right indicates frequencies (%) of mutant (base-substituted) alleles and - stands for the absence of nucleotides at corresponding positions (deletion); Numerals on the left indicate Dmd mutant mouse numbers),

[0132] FIG. 1c shows Sanger sequencing chromatograms of DNA from wild-type and Dmd mutant mice D108 (the arrow indicates the substituted nucleotide, showing the conversion of a Gln codon to a stop codon by base substitution),

[0133] FIG. 1d shows histological analysis (fluorescence analysis) results of tibialis anterior (TA) muscles from wild-type and Dmd mutant mice D108 (Laminin: control; muscles were dissected from 4-week-old wild-type or Dmd mutant mice D108 and frozen in liquid nitrogen-cooled isopentane; scale bars: 50 m).

[0134] FIG. 1e summarizes procedures of inducing base substitutions at the target site in the Dmd gene and the results.

[0135] FIGS. 2a to 2e pertain to the generation of albinism mice by cytidine deaminase-mediated base editing,

[0136] FIG. 2a shows the nucleotide sequence at the target site in the tyrosinase gene (Tyr) (the PAM sequence (NGG) and the sgRNA target sequence are shown in blue and black, respectively; the nucleotides substituted by cytidine deaminase-mediated base editing is shown in red),

[0137] FIG. 2b shows alignments of nucleotide sequences at the target site in the Tyr gene from newborn pups that developed after base editor 3 (BE3) (rAPOBEC1-nCas9-UGI) RNP (complex of BE3 and sgRNA hybridizable with the target sequence of the Tyr gene of FIG. 2a) was introduced to mouse zygotes by electroporation (Wt, wild-type; the target sequence is underlined; the PAM sequence and substitutions are shown in blue and red, respectively; the column on the right indicates frequencies (%) of mutant (base-substituted) alleles and - stands for the absence of nucleotides at corresponding positions (deletion); Numerals on the left indicate Tyr mutant mouse numbers),

[0138] FIG. 2c shows Sanger sequencing chromatograms of DNA from wild-type and Tyr mutant mice T113 and T114 (the arrows indicate the substituted nucleotides, showing the conversion of a Gln codon to a stop codon by base substitution),

[0139] FIG. 2d shows an albino phenotype in the eyes of Tyr mutant newborn pups that developed after electroporation of the BE3 RNP (T113 and T114 indicated by arrows),

[0140] FIG. 2e summarizes procedures of inducing base substitutions at the target site in the Tyr gene and the results.

[0141] FIGS. 3a and 3b are alignments of the nucleotide sequences at the target sites in the target genes (Dmd and Tyr) of blastocysts that developed after microinjection of BE3-encoding mRNA and sgRNA into mouse zygotes, showing the induction of targeted mutations in mouse embryos by microinjection of BE3(deaminase-Cas9) mRNA and sgRNA thereinto (3a: Dmd mutation result; 3b: Tyr mutation result; Wt, wild-type; the target sequences are underlined; the PAM sequences and substitutions are shown in blue and red, respectively; the column on the right indicates frequencies (%) of mutant (base-substituted) alleles and - stands for the absence of nucleotides at corresponding positions (deletion); Numerals on the left indicate Tyr mutant mouse numbers),

[0142] FIG. 3 shows Sanger sequencing chromatograms of DNA from wild-type and Dmd mutant mice (the arrows indicate substituted nucleotides).

[0143] FIG. 4 is a graph of results after testing whether or not Dmd mutations are generated at potential off-target sites, showing that no off-target mutations are detectably induced at the off-target sites (Targeted deep sequencing was used to measure mutant ratios (base editing efficiencies) (%) at potential off-target sites in Dmd mutant mice (n=3); mismatched nucleotides and PAM sequences are shown in red and blue, respectively).

[0144] FIG. 5 is a graph of results after testing whether or not Tyr mutations are generated at potential off-target sites, showing that no off-target mutations are detectably induced at the off-target sites (Targeted deep sequencing was used to measure mutant ratios (base editing efficiencies) (%) at potential off-target sites in Tyr mutant mice (n=2); mismatched nucleotides and PAM sequences are shown in red and blue, respectively).

[0145] FIG. 6 is a cleavage map of the pCMV-BE3 vector.

[0146] FIG. 7 is a cleavage map of the pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS vector.

MODE FOR CARRYING OUT THE INVENTION

[0147] Hereafter, the present disclosure will be described in detail by examples. The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

Example 1: Preparation of BE3 mRNA

[0148] After being isolated by digestion from pCMV-BE3 (Addgene; cat. #73021; FIG. 6), rAPOBEC1-XTEN (linker) and UGI (uracil DNA glycosylase inhibitor) were inserted into the pET-nCas9 (D10A)-NLS vector (see Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24, 132-141 (2014)) to construct pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS (SEQ ID NO: 7; FIG. 7) which was then used as a BE3 mRNA template.

[0149] Sequences of individual regions in pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS (SEQ ID NO: 7) are summarized as follows:

[0150] His x6: SEQ ID NO: 8;

[0151] rAPOBEC1: SEQ ID NO: 9;

[0152] XTEN (linker): SEQ ID NO: 10;

[0153] nCas9 (D10A): SEQ ID NO: 11;

TABLE-US-00001 Linker: (SEQIDNO:14) TCTGGTGGTTCT

[0154] UGI: SEQ ID NO: 12;

TABLE-US-00002 Linker: (SEQIDNO:14) TCTGGTGGTTCT

[0155] NLS: SEQ ID NO: 13.

[0156] PCR was performed on the pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS vector with the aid of Phusion High-Fidelity DNA Polymerase (Thermo Scientific) in the presence of primers (F: 5-GGT GAT GTC GGC GAT ATA GG-3, R: 5-CCC CAA GGG GTT ATG CTA GT-3) to prepare the mRNA temperature. From the prepared mRNA template, BE3 mRNA was synthesized using an in vitro RNA transcription kit (mMESSAGE mMACHINE T7 Ultra kit, Ambion), followed by purification with MEGAclear kit (Ambion).

Example 2: Preparation of sgRNA

[0157] A dystrophin gene Dmd and a tyrosinase gene Tyr targeted guide RNA (sgRNA) having the following nucleotide sequence were synthesized and used in subsequent experiments:

TABLE-US-00003 5-(targetsequence)-(GUUUUAGAGCUA;SEQIDNO:1)- (nucleotidelinker)- (UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGC;SEQIDNO:3)-3

[0158] (the target sequence is the same sequence as the underlined nucleotide sequence in FIG. 1a (Dmd) or FIG. 2a (Tyr), with the exception that T is converted to U, and

[0159] the nucleotide linker has the nucleotide sequence of GAAA).

[0160] The sgRNA was constructed by in vitro transcription using T7 RNA polymerase (see Cho, S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230-232 (2013)).

Example 3: Preparation of Ribonucleoproteins (RNPs)

[0161] Rosetta competent cells (EMD Millipore) were transformed with the pET28-Hisx6-rAPOBEC1-XTEN-nCas9(D10A)-UGI-NLS (BE3) expression vector prepared in Reference Example 1 and then incubated with 0.5 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) at 18 C. for 12 to 14 hours to induce expression. Following protein expression, bacterial cells were harvested by centrifugation and the cell pellet was lysed by sonication in a lysis buffer [50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1% Triton X-100, 1 mM PMSF, 1 mM DTT, and 1 mg/ml lysozyme].

[0162] The cell lysate thus obtained was subjected to centrifugation at 5,251g for 30 min to remove cell debris. The soluble lysate was incubated with Ni-NTA beads (Qiagen) at 4 C. for 1 hr. Subsequently, the Ni-NTA beads were washed three times with wash buffer [50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 20 mM imidazole], followed by eluting BE3 protein with elution buffer [50 mM Tris-HCl (pH 7.6), 150-500 mM NaCl, 10-25% glycerol, and 0.2 M imidazole]. The purified BE3 protein was dialyzed against storage buffer [20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol] and concentrated using Ultracell 100K cellulose column (Millipore). The purity of the protein was analyzed by SDS-PAGE. SgRNA was prepared by in vitro transcription using T7 RNA polymerase as described in Example 3.

Example 4: Preparation of Animal

[0163] Experiments on mice were conducted after approval by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University. Mice were maintained in a SPF (specific pathogen-free) condition, with a 12/12 hrs light/dark cycle. C57BL/6J and ICR mice were used as an embryo donor and a surrogate mother.

Example 5: Microinjection and Electroporation into Mouse Zygote

[0164] Superovulation, embryo collection, microinjection, and electroporation were performed with reference to Hur, J. K. et al., Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34, 807-808 (2016).

[0165] For microinjection, a solution containing a complex of BE3 mRNA (10 ng/ul) and sgRNA (100 ng/ul) was diluted in DEPC-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4) (see Sung, Y. H. et al. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 24, 125-131 (2014)) and then injected into pronuclei of one-cell stage zygote at fertilization with the aid of Nikon ECLIPSE Ti micromanipulator and FemtoJet 4i microinjector (Eppendorf).

[0166] For electroporation, a BE3-sgRNA RNP complex was introduced to one-cell mouse embryos by electroporation using a NEPA 21 electroporator (NEPA GENE Co. Ltd.) including a glass chamber filled with 100 l of opti-MEM (Thermo Fisher Scientific) containing the BE3-sgRNA RNP complex (10 g/100 l and 6.5 g/100 l, respectively) (see Hur, J. K. et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34, 807-808 (2016)).

[0167] After BE3 RNP or mRNA transfer, the embryos were cultured in microdrops of KSOM+AA (Millipore) at 37 C. for 4 days under a humidified condition of 5% CO2. Two-cell stage embryos were transplanted to the oviduct of a 0.5-dpc pseudo pregnant surrogate mother.

[0168] The procedure is summarized in the following diagram:

TABLE-US-00004 Cytidine deaminase-mediated base editing in mice No. of No. of 2-cell No. of Mutant ratio (%) examined stage No. of No. of transferred offsprings (No. of mutant/total Target gene Methods Method embryos embryos (%) blastocysts (%) embryos (%) blastocysts or offsprings) Dmd mRNA + sgRNA Microinjection 33 30 (91%) 15 (50%) 66 11 67% (16/24) mRNA + sgRNA Electroporation 60 56 (93%) 30 (54%) NA NA 10% (3/30) RNP Electroporation 55 52 (95%) 16 (31%) NA NA 75% (12/16) Tyr mRNA + sgRNA Microinjection 23 21 (91%) 10 (48%) NA NA 90% (9/10) mRNA + sgRNA Electroporation 65 54 (83%) 16 (30%) NA NA 13% (2/16) RNP Electroporation 53 47 (89%) 14 (30%) NA NA 77% (10/13) NA: not applicable a: Calculated from the number of examined embryos b: Calcuated from the number of developed 2-cell stage embryos

Example 6: Genotyping

[0169] For PCR genotyping, genomic DNA was extracted from the blastula stage embryo obtained from the embryo transplanted to the oviduct in Reference Example 4 or from an ear clip of the newborn pups and subjected to targeted deep sequencing and Sanger sequencing.

Example 7: Targeted Deep Sequencing

[0170] In this Example, nucleotide sequences were analyzed using targeted deep sequencing as follows.

[0171] Target sites or off-target sites were amplified from the genomic DNA extracted in Reference Example 5 with the aid of Phusion polymerase (Thermo Fisher Scientific). Paired-end sequencing of the PCR amplicons was performed using Illumina MiSeq (LAS, Inc. (South Korea) commissioned to perform). Primers used in the amplification of off-target sites are given in Tables 1 and 2, blow.

TABLE-US-00005 TABLE1 Primersusedfortargeteddeepsequencingatpotentialoff-targetsites ofDmdtargetedsgRNA 1stPCR 2ndPCR No. Forward(5-3) Reverse(5-3) Forward(5-3) Reverse(5-3) Dmd- TTTTTGCTCC TTATGTGGCCT ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT1 TTACAAACAA TGCTCATTG CGACGCTCTTCCGAT ACGTGTGCTCTTCCG GG CTTCAGAGGAGCTAC ATCTCAGTGAGTCATT AAGCAAGG GCATCCATC Dmd- ACCCCAAAAT TGAGGTCAGG ACACTCTTTCCCTAC GTGACTGGAGTTCAG OT2 TCACCAGAGA GATGGTGATT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTTGAAAGGATACATG ATCTAATCCTGACAAT GGCTGA CCATGAACA Dmd- TTTGAGCCTC TCAGCCTCTTC ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT3 TGGGAACATT TCCACTTTTT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTATCTATGCTGCCGA ATCTCCCAAATTGTAT TGATCC AACTGAAGCAG Dmd- TGAAGTCCTA TGGCATTGCAT ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT4 GAAAACAAAA TGAATCTGT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG GCA CTAAAGGATAAACAG ATCTTGGGATATCTTT GCTGAGAAAAA CCAATTTCTGA Dmd- GCTTTCTAAA CACCTGCCAA ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT5 GCCTTTTTAG GTGTGGTATG CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTTTT CTTTCTGGCAGTTTAC ATCTTGAAAGTGACAG CCAAGG CAATTCCATT Dmd- CAACCCATAT TGCCAATTGCC ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT6 ATATTTTGGC CTTTCTATC CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CAGT CTGCACCTAATCAGT ATCTCCATGAGCATG GGCCTTT GGAAATCTT Dmd- TTTCAGGGAA TGTGTGCAATA ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT7 AGGTGACAG AACCACAGTGA CGACGCTCTTCCGAT ACGTGTGCTCTTCCG G CTTTTCAGGGAAAGG ATCTGCAGTTTCACGT TGACAGG CTGGGAGT Dmd- CATCCAAAGT GTCTGGCCCA ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT8 GGCTTGAACA AACAGAATGT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTGCACCTCCCATAA ATCTCATGAATCTTCC CCGTAAA CACAAGGAA Dmd- GCACCTAGAT AATGCCAAATG ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT9 TTTGGCCATC CATTGAAGG CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTTTAAAGGCATGCAC ATCTAATGCCAAATGC AACCAC ATTGAAGG Dmd- TGCAAGTTGT AGGCAAGGTG ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT10 CTTCCGACTG AGGACTCAGA CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTGCCAGACATGCAC ATCTCCATTCTCACAG ACACATA TTATCCCAAA Dmd- CGAGAGGTTG CTCCTGAGGG ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT11 AGACCTGGAG TAGGGAGCTT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTAACCACACTACTGC ATCTACCCCTGAGAAA TCTCATGC TGAACACG Dmd- GTTTGGCGTG GCACATCTTCC ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT12 GGATATGACT ATGTGCTGT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG CTACTCCAACCAACA ATCTTAGGCTGTGGT GGACGAT GATGGCTTT Dmd- AGAAGAGGG GGTCTTAGCCT ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT13 CCATGAGTCA CCAGCCTCT CGACGCTCTTCCGAT ACGTGTGCTCTTCCG A CTAAGATAACGCCATA ATCTCCCTTCTTTTCC GCTGCAC AGCTCCTT Dmd- CCAGTCGTTA GGGTGACCTT ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT14 GGCCTGTGA GAATTCCTGA CGACGCTCTTCCGAT ACGTGTGCTCTTCCG G CTTCAGGAGCGCACT ATCTAGGCATGTACCA AGAACCT TGACCACA Dmd- GAGAACGAGT CCCATGTATTT ACACTCTTTCCCTACA GTGACTGGAGTTCAG OT15 GCCAAAGGA TCCCATTGC CGACGCTCTTCCGAT ACGTGTGCTCTTCCG G CTTAACACATGGTTGG ATCTCTAGTTATGAGG CTCCTT GAACAGTTGCT

TABLE-US-00006 TABLE2 Primersusedfortargeteddeepsequencingatpotentialoff-targetsitesofTyr targetedsgRNA 1stPCR 2ndPCR No. Forward(5-3) Reverse(5-3) Forward(5-3) Reverse(5-3) Tyr-OT1 GCATGAGCAC AGATGCCTGCT ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA ACACTGAAAT CTGTCTTTC GACGCTCTTCCGATCTA CGTGTGCTCTTCCGAT ATAA GTCCCAAGTACCCTACT CTAAACAAGAGCTCACA AC TTATTGG Tyr-OT2 AGTGGTTGGC GCATGGTATGT ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA TTCTCTCTTAT ACTCCCTGTT GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT C GCTTCTCTCTTATCCCA CTCCCACAGACAGTAA CTCATATC GAAGTTCA Tyr-OT3 CATGTGTCTT TCTGTGGCCTA ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA CCTGGCTATC GAGGAGTAAT GACGCTCTTCCGATCTA CGTGTGCTCTTCCGAT TT CTTGTGCTATGCATTGG CTATCAAGAGAAGGCA TAGA GCACATAG Tyr-OT4 TTGTCTTCCT AGATTATGCCC ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA GTGTCTGCCT AAGGGGTTT GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT TA CCTATCCTATCCCCCTC CTGGGACTAAACCACC TGC ACCAGA Tyr-OT5 TGGCCAGAAG AGGGTTTGCAA ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA ACTAGGATGG CTCCATAGG GACGCTCTTCCGATCTT CGTGTGCTCTTCCGAT CTTTTCCCAGTTTCCTT CTTGTAGCAGAGAATG TCC GCCTTG Tyr-OT6 TCACACCAGC GAGAAAGGAC ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA TTGCCATT CAAAGGAGTT GACGCTCTTCCGATCTA CGTGTGCTCTTCCGAT GA CACCAGCTTGCCATTCT CTGGGAGAGGTCCTTG T ATCCTAT Tyr-OT7 CCAACCAGAA CTCTTCCTCTT ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA CCACCAGAAT CCTCTTCCTCT GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT CAGAATTCCCAGGGAC CTCTCCCTCCTCCTGAT TAAGC TCTATGA Tyr-OT8 CAGTTTCGGT CAATTGATAGT ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA AGCCTTGACT GGCTGCCTAG GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT TA A CAGTGCGATAACCCTT CTGGATTGCGAAACAG CTTGT TGGATCT Tyr-OT9 GGGAAATAGT GAGACTGGAA ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA AAGTAACAAG CAGCAAACAC GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT GAGAA CCACTTGTATGAGGGT CTAAATGCCCATGCAG GTTTCT CTCT Tyr- TTTTGTTGTCA TCCAGGGATTT ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT10 GCTGGCTTG TGTGTTGGT GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT CCCTATCCCATCCACTT CTAAGCCATCAACAAAG TCC GATGG Tyr- CTTTCCCAGT GAGCCTTACAA ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT11 GCCACCTAAA ATACAGAGATG GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT GA CTTTCCCAGTGCCACCT CTGACTAAGCCATCAAC AAA CAAGGA Tyr- CAAGGCCGG CCTCCGCTAAC ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT12 AGAGTTTACT ACAACATACA GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT AAG GCCATCTAAGTAGGAA CTCAGCAGGGAGTAGG GGCTAGA ATGTAAGTA Tyr- TACTCTGCTG TGTGTGTGTGT ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT13 CAAGAGGATT GTGTGTGT GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT TC GATGGATGCCTACCTG CTGAAGTAGACAGCAT ACAAA AGAGTACAGAAG Tyr- AAAGGACTGA ACGTCCAGGA ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT14 AGGAGTTGAA AGTTCTCTTTA GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT GG TG CAAAGAGCTCACAGGG CTCCTGCTTCTATGAGG ACTAAA GTGTTC Tyr- GGCCCTGTCT GGATATAACTC ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT15 ATTTACTAGA ACAGACCTCAA GACGCTCTTCCGATCT CGTGTGCTCTTCCGAT GTTG GAA GGCTCCACATTTCCATT CTGGATATAACTCACAG CATTC ACCTCAAGAA Tyr- AGGAAGGAAG GTGAGGCAAA ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT16 AAACTGAAAC CCCACAAGTA GACGCTCTTCCGATCTA CGTGTGCTCTTCCGAT CA AAGAGGCCCAAGGATC CTATACATCTGACCCAC AC CTTGC Tyr- ATTGTTGTGT GCTCTATTACC ACACTCTTTCCCTACAC GTGACTGGAGTTCAGA OT17 CTTCTGCCCT CAGTTCCTTCC GACGCTCTTCCGATCTA CGTGTGCTCTTCCGAT A GTCACAACTGTAGGCA CTGTTCCTTCCTCTACC CATT AGAAAGC

Example 8: Immunofluorescent Staining

[0172] Tibialis anterior (TA) muscle sections resected from the mouse were immunostained with a laminin or dystrophin antibody. Laminin was detected using a 1:500 dilution of a rabbit polyclonal antibody (abcam, ab11575) and a 1:1000 dilution of an Alexa Fluor 568 anti-rabbit secondary antibody (Thermo Fisher Scientific) sequentially. For dystrophin detection, a 1:500 dilution of rabbit polyclonal antibody (abcam, ab15277) and a 1:1000 dilution of an Alexa Fluor 488 anti-rabbit secondary antibody (Thermo Fisher Scientific) were used sequentially. The immunofluorescently stained sections were observed with a Leica DM14000 B fluorescence microscope.

Example 9: Sequencing of Mouse Embryo Having Mutation Induced with BE3

[0173] As described in Examples 1-5, Base Editor 3 (BE3) (rAPOBEC1-nCas9-UGI) was introduced to mouse embryonic cells by microinjection to induce a point mutation in each of the dystrophin-encoding gene Dmd and the tyrosinase-encoding gene Tyr.

[0174] As shown in FIGS. 1a (Dmd) and 2a (Tyr), the generation of a stop codon would be predicted by single base substitution (C.fwdarw.T) at a target site (underlined sequence sites of upper sequences in FIGS. 1a and 2a) in each gene (in FIGS. 1a and 2a, single base substitutions occurred on lower nucleotide sequences, with substituted (C.fwdarw.T) bases appearing red).

[0175] FIGS. 1e and 2e show summaries of procedures of target-specific single base substitution (microinjection or electroporation) and results thereof. As shown in FIGS. 1e and 2e, embryonic mutations were observed at target sites in Dmd and Tyr genes at frequencies of 73% (11 of 15 for Dmd) and 100% (10 of 10 for Tyr), respectively.

[0176] In addition, nucleotide sequences of target sites in mouse embryos in which mutations had been induced by microinjecting BE3 mRNA and sgRNA were identified by targeted deep sequencing. In greater detail, target-specific mutation was induced by microinjecting mouse BE3 (rAPOBEC1-nCas9-UGI) mRNA and sgRNA into mouse embryos. For this, BE3-encoding mRNA and sgRNA were microinjected to mouse zygotes, and then nucleotide sequences of target sites in the target genes (Dmd and Tyr) in the resulting blastocysts were aligned.

TABLE-US-00007 [MutationresultsofDmdtargetedmRNAmicroinjection] Sequence Frequency(%) Wt ACAGCAATTAAAAGCCAGTTAAAAATTTGTAAGG (SEQIDNO:15) #64 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 94 #65 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 53 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 38 #66 ACAGCAATTAAAAGACAGTTAAAAATTTGTAAGG 55 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 34 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 6 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 3 #67 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 16 #68 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 97 #72 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 71 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 25 #79 ACAGCAATTAAAAGGTAGTTAAAAATTTGTAAGG 99 #80 ACAGCAATTAAAAGTAAGTTAAAAATTTGTAAGG 39 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 20 ACAGCAA---------------AAATTTGTAAGG 40(-15bp) #87 ACAGCAATTAAAAGCAAGTTAAAAATTTGTAAGG 84 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 12 #88 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 77 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 22 #95 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 54 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 45 [MutationresultsofTyrtargetedmRNAmicroinjection] Sequence Frequency(%) Wt GCACCATCTGGACCTCAGTTCCCCTTCAAAGGGG (SEQIDNO:17) #47 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 73 #48 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 99 #49 GCACCATCTGGACCTTAGTTTCCCTTCAAAGGGG 49 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 45 #50 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 99 #51 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 56 GCACCATCTGGACCTCAGTTCCCTTTCAAAGGGG 14 GCACCATCTGGACCTCA--------TCAAAGGGG 25(-8bp) #52 GCACCATCTGGACCTTAGTTCCC-TTCAAAGGGG 100(-1bp) #53 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 26 GCACCATCTGGACCTAAGTTCCCCTTCAAAGGGG 23 #54 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 57 #55 GCACCATCT---------------------GGGG 16(-21bp) GCACCATCTGGACCTAAGTTCCCCTTCAAAGGGG 15 GCACCATCTGGATCTTAGTTCCCCTTCAAAGGGG 14 GCACCATCTGGACCTTAGTTACCCTTCAAAGGGG 11 #56 GCACCATCTGGACCTTAGTTCCCCTTCAAAGGGG 86 (Wt, wild-type; the target sequence is underlined; the PAM sequence (NGG) is shown in bold; substituted bases are in bold and underlined; the column on the right indicate frequencies (%) of mutant (base substituted) alleles and -stands for absence of nucleotides at corresponding positions (deletion); numerals on the left are mutated mouse embryonic cell numbers)

[0177] As is understood from the aligned sequences, C.fwdarw.T base substitution is a predominant mutation pattern at both the target sites in the two genes (Dmd and Tyr).

Example 10: Identification and Induction of Mutation in Mouse Subject by Microinjection of BE3 and Dmd Targeted sgRNA

[0178] After microinjection of BE3 mRNA and Dmd targeted sgRNA thereinto, mouse embryos were transplanted to the oviduct of foster surrogate mothers (see Example 5) to give mutant newborn pups having point mutation on the Dmd gene thereof (F0).

[0179] FIGS. 1b and 3 show analysis results of nucleotide sequences of the target site in the target gene (Dmd) of newborn pups developed after BE3 (rAPOBEC1-nCas9-UGI)-encoding mRNA and sgRNA hybridizable with the nucleotide sequence of the target site in the Dmd gene have been injected into moue zygotes. As can be seen in FIG. 1b, when point mutation was induced in the Dmd gene, five (D102, D103, D107, D108, and D109) among a total of nine mice had mutation at the target site in the Dmd gene. Of the five mutant mice, three subjects (D102, D103, and D108) were found to have one or two mutant allele genes and lack wild-type allelomorphic characteristics. The other two mutant mice (D107 and D109) retained wild-type allele genes in a mosaic pattern at a frequency of 10%. In addition, as can be seen FIGS. 1b and 3, the mutant mouse D109 exhibited 20-base pair (bp) deletion other than point mutation, demonstrating that the Cas9 nickase included in BE3 retains the activity of inducing indels at the target site.

[0180] FIG. 1c shows Sanger sequencing chromatograms of the target site in the target gene of wild-type mouse and Dmd mutant mouse D108. As shown in FIG. 1c, the mutant F0 mouse D108, which lacks a wild-type allele gene, had an early stop codon (TAG) introduced by single base substitution (C.fwdarw.T) to the Dmd gene thereof.

[0181] FIG. 1d shows images of immunofluorescent stained TA muscle sections from the wild-type mouse and the Dmd mutant mouse D108 (see Example 8), exhibiting that dystrophin was nearly not expressed in the muscle of the mutant subject (D108). The result implies that the Dmd gene was successfully knocked down by the injection (microinjection of BE3 mRNA and Dmd targeted sgRNA.

Example 11: Identification and Induction of Mutation in Mouse Embryo by Electroporation of RNA Comprising BE3 and sgRNA

[0182] BE3 ribonucleproteins (RNPs), prepared in Example 3, including a mixture (rAPOBEC1-nCas9(D10A)-UGI RNP) of the recombinant BE3 protein and the in-vitro transcribed sgRNA were transferred to mouse embryos by electroporation (see Example 5). Four days after electroporation, nucleotide sequences of target sites in the target genes (Dmd and Tyr) of the mouse embryos were analyzed and the results are given as follows:

TABLE-US-00008 [Dmd,RNPelectroporationmutatinresult] Sequence Frequency(%) Wt ACAGCAATTAAAAGCCAGTTAAAAATTTGTAAGG (SEQIDNO:15) #17 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 85 #18 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 66 ACAGCAATTAAAAGCAAGTTAAAAATTTGTAAGG 18 #19 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 64 ACAGCAATTAAAAGCAAGTTAAAAATTTGTAAGG 24 #20 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 57 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 30 #21 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 100 #22 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 96 #23 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 42 #24 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 51 #25 ACAGCAATTAAAAGTTAGTTAAAAATTTGTAAGG 68 ACAGCAATTAAAAGTCAGTTAAAAATTTGTAAGG 8 #26 ACAGCAATTAAAAGCCAGTT--------GTAAGG 98(-8bp) #28 ACAGCAATTAAAAGTAAGTTAAAAATTTGTAAGG 100 #31 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 77 #32 ACAGCAATTAAAAGCTAGTTAAAAATTTGTAAGG 100 [Tyr,RNPelectroporationmutationresult] Sequence Frequency(%) Wt GCACCATCTGGACCTCAGTTCCCC-TTCAAAGGGGTGG (SEQIDNO:17) #83 GCACCATCTGGACCTAAGTTCCCC-TTCAAAGGGGTGG 25 GCACCATCTGGACCTCAGTTACCC-TTCAAAGGGGTGG 18 GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 14 #34 GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 54 GCACCATCTGGACCTGAGTTCCCC-TTCAAAGGGGTGG 45 #36 GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 48 #37 GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 56 #38 GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 94 #40 GCACCATCTGGACCTGAGTTCCCC-TTCAAAGGGGTGG 59 GC-----------------------TTCAAAGGGGTGG 21(-22bp) GCACCATCTGGACCTCAGTTCCCC-TTCAAAGGGGTGG 14 #41 GCACCATCTGGACCTTAGTTTCCC-TTCAAAGGGGTGG 44 #42 G------------------------------------G 35(-35bp) #44 GCACCATCTGGACCTTAGTTCCCCTTTTAAAGGGGTGG 25(+1bp) GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 9 GCACCATCTGGACCTCAGTTCTCC-TTCAAAGGGGTGG 3 #45 GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 55 #46 GCACCATCTGGACCTCAG-----------------TGG 76(-16bp) GCACCATCTGGACCTTAGTTCCCC-TTCAAAGGGGTGG 23 (Wt, wild-type; the target sequence is underlined; the PAM sequence (NGG) is shown in bold; substituted bases are in bold and underlined; the column on the right indicate frequencies (%) of mutant (base substituted) alleles and stands for absence of nucleotides at corresponding positions (deletion); numerals on the left are mutated mouse numbers)

[0183] As understood from the results and FIGS. 1e and 2e, the electroporation induced mutations at the Dmd and Tyr target sites in the blastocyst embryos at frequencies of 81% (13 of 16 for Dmd) and 85% (11 of 13 for Tyr).

Example 12: Identification and Induction of Mutation in Mouse Subject by Electroporation of BE3 and Tyr Targeted sgRNA

[0184] After electroporation of BE3 and Tyr targeted sgRNA thereinto, mouse embryos were transplanted to the oviduct of surrogate mothers (see Example 5) to give mice having point mutation on the Tyr gene thereof (F0).

[0185] FIG. 2b shows alignment of nucleotide sequences at the target site in Tyr genes of the mutant newborn pups thus obtained. As shown in FIG. 2b, various mutations were induced at the target site in the Tyr gene of all of the seven mutant newborn pups (T110, T111, T112, T113, T114, T117, and T118).

[0186] FIG. 2c shows Sanger sequencing chromatograms of the target site in the target gene of wild-type mouse and mutant newborn pups (T113 and T114). As seen, the mutant newborn pups had a stop codon (TAG) introduced by single base substitution (C.fwdarw.T) to the target site.

[0187] FIG. 2d shows phenotypes of the eyes of the mutant newborn pups, exhibiting ocular albinism in the mutant mice (T113 and T114). The result implies that the Tyr gene was successfully knocked down by the introduction (electroporation) of RNP of BE3 mRNA and Tyr targeted sgRNA.

Example 13: Assay for Off-Target Effect

[0188] In order to assay off-target effects of BE3, potential off-target sites having up to 3-nucleotide mismatches were found in the moue genome by using the Cas-OFFinder (http://www.rgenome.net/cas-offinder/) and genomic DNA isolated from the mutant newborn pups were analyzed using targeted deep sequencing.

[0189] sgRNA sequence and primer sequences used in targeted deep sequencing are summarized in Table 3, below.

TABLE-US-00009 TABLE3 sgRNAsequence Dmd AAGCCAGTTAAAAATTTGTAAGG(SEQIDNO:19) Tyr ACCTCAGTTCCCCTTCAAAGGGG(SEQIDNO:35) sgRNAprimerforT7invitrotranscription(5-3) Dmd-F GAAATTAATACGACTCACTATAGAAGCCAGTTAAAA ATTTGTAGTTTTAGAGCTAGAAATAGCAAG(SEQID NO:53) Tyr-F GAAATTAATACGACTCACTATAGACCTCAGTTCCCC TTCAAAGGTTTTAGAGCTAGAAATAGCAAG(SEQID NO:54) R AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAAC GGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC (SEQIDNO:55) Targeteddeepsequencingprimer(5-3) Dmd-1stPCR-F GCTAGAGTATCAAACCAACATCATTAC(SEQIDNO: 56) Dmd-1stPCR-R TGCTTCCTATCTCACCCATCT(SEQIDNO:57) Dmd-2nd/adaptor ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCT PCR-F ACAACAATTGGAACAGATGAC(SEQIDNO:58) Dmd-2nd/adaptor GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTC PCR-R TTCACTGTACAGAGCTCAATG(SEQIDNO:59) Tyr-1stPCR-F TGTATTGCCTTCTGTGGAGTT(SEQIDNO:60) Tyr-1stPCR-R GGTGTTGACCCATTGTTCATTT(SEQIDNO:61) Tyr-2nd/adaptor ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGA PCR-F GTTTCCAGATCTCTGATGG(SEQIDNO:62) Tyr-2nd/adaptor GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGC PCR-R ACTGGCAGGTCCTATTAT(SEQIDNO:63)

[0190] The off-target sites identified in the mouse genome and the targeted deep sequencing results are given in Tables 4 (Dmd) and 5 (Tyr) and FIGS. 4 (Dmd) and 5 (Tyr).

TABLE-US-00010 TABLE4 Off-targetsitesforDmdtargetsiteinmousegenome(Dmd-On:on-targetsiteof Dmd;Dmd-OT:off-targetsiteofDmd) Chromo- No. Gene Sequence some Position Direction Dmd- Dmd Exon AAGCCAGTTAAAAATTTG chrX 83781748 + On TAAGG(SEQIDNO:19) Dmd- Atp11c Intron AAGCCAGTTAgAAgTTTG chrX 60354718 + OT1 TAAGG(SEQIDNO:20) Dmd- Intergenic AAGCaAGTgtAAAATTTGT chr3 123915964 - OT2 region ATGG(SEQIDNO:21) Dmd- Ptprd Intron AAtaCAGTTAAtAATTTGTA chr4 78045958 + OT3 AGG(SEQIDNO:22) Dmd- Cfh Intron AAGCaAGTaAAAcATTTGT chr1 139933967 - OT4 ATGG(SEQIDNO:23) Dmd- Sos1 Intron ttGCCAGTTtAAAATTTGTA chr17 80462531 - OT5 AGG(SEQIDNO:24) Dmd- Intergenic AAGCaAtTgAAAAATTTGT chr17 81921018 + OT6 region ATGG(SEQIDNO:25) Dmd- Grid2 Intron AAGCCAGaaAcAAATTTG chr6 64297703 - OT7 TAGGG(SEQIDNO:26) Dmd- Intergenic AAGCtAGTggAAAATTTGT chr6 147759582 - OT8 region ACGG(SEQIDNO:27) Dmd- Intergenic AActCAaTTAAAAATTTGT chr14 26539529 - OT9 region ATGG(SEQIDNO:28) Dmd- Spag9 Intron AAGCCAGagAAtAATTTGT chr11 94052933 + OT10 AGGG(SEQIDNO:29) Dmd- Tle1 Intron AAaCCAGTTAAAtATTTcT chr4 72159600 + OT11 AAGG(SEQIDNO:30) Dmd- RNf114b Intron AAGCCAtTTAAAAATTTGa chr13 47235657 - OT12 gTGG(SEQIDNO:31) Dmd- Tnfrsf21 Intron AAcCCAGTTAgAAATTTtT chr17 43052831 + OT13 ATGG(SEQIDNO:32) Dmd- Aak1 Intron AAGaCAGaTAAAAATTTG chr6 86907351 - OT14 gAGGG(SEQIDNO:33) Dmd- Gpm6b 3UTR AgGCCAGaTAAAAATTTG chrX 166385994 - OT15 aAGGG(SEQIDNO:34)

TABLE-US-00011 TABLE5 Off-targetsiteforTyrtargetsiteinmousegenome(Tyr-On:on-targetsiteofTyr; Tyr-OT:off-targetsiteofTyr) Chromo- No. Gene Sequence some Position Direction Tyr- Tyr Exon ACCTCAGTTCCCCTTCAA chr7 87493130 - On AGGGG(SEQIDNO:35) Tyr- Zmat4 Intron cCCTCAGTTCCaCTTCAg chr8 23975596 - OT1 AGAGG(SEQIDNO:36) Tyr- Intergenic ACCTCAcTTgCCCTTCtAA chr8 102059942 + OT2 GTGG(SEQIDNO:37) Tyr- Il2 Intron ACCTCAGTcCCCCTTtAcA chr3 37123254 + OT3 GAGG(SEQIDNO:38) Tyr- Intergenic ACCTCAGTTCCCCTaCAct chr3 9150768 - OT4 GGGG(SEQIDNO:39) Tyr- Intergenic ACCTCAGTTCCCCTaCAct chr3 81773804 - OT5 GGGG(SEQIDNO:40) Tyr- Intergenic tCCTCAGTTCCCCTTCAct chr7 11778250 + OT6 GGGG(SEQIDNO:41) Tyr- Intergenic cCCTCAGTTCCCCTaCAc chr4 47766122 - OT7 AGAGG(SEQIDNO:42) Tyr- Intergenic ACCTCAGTTtCCCTTCcAg chr4 54553337 - OT8 GAGG(SEQIDNO:43) Tyr- 2810429I04Rik Intron cCCTCAGTTCCCCTTCAct chr13 3491261 + OT9 GGGG(SEQIDNO:44) Tyr- Intergenic cCCTCAGTTCCCCTaCAc chr13 74957186 + OT10 AGGGG(SEQIDNO:45) Tyr- MGP_C57BL6NJ_ cCCTCAGTTCCCCTaCAc chr2 79031825 + OT11 G0001126 AGGGG(SEQIDNO:46) Tyr- Rai14 Intron ACCTCAGTTtCCCcTCAAA chr15 10596653 + OT12 aTGG(SEQIDNO:47) Tyr- Intergenic ACCTCAGTTgtCCTTCAAA chr6 107384348 - OT13 cAGG(SEQIDNO:48) Tyr- D6Ertd474e Intron cCCTCAGTTCCCCTaCAAt chr6 143247456 - OT14 GGGG(SEQIDNO:49) Tyr- Intergenic AaCTCtGTTCCCCTTCtAA chr18 23151245 + OT15 GTGG(SEQIDNO:50) Tyr- Intergenic AtCTCAGTTtCCCTTCAcA chr11 71708976 - OT16 GGGG(SEQIDNO:51) Tyr- Foxk2 Intron ACCTtAGTTCCCtTTCAAA chr11 121271649 + OT17 cTGG(SEQIDNO:52)

[0191] (In Tables 5 and 6, mismatched nucleotides in off-target sites with the on-target sequence are represented in lower cases: NGG at the 3 end accounts for the PAM sequence)

[0192] As can be seen in Tables 4 and 5 and FIGS. 4 and 5, the on-target sites used in this assay were observed not to induce significant off-target mutation, demonstrating that the BE3 system targeting the on-target sites is significantly specific for the targets in vivo.