CN106939303B

CN106939303B - Cas9 nuclease R919P and application thereof

Info

Publication number: CN106939303B
Application number: CN201710343933.4A
Authority: CN
Inventors: 吴强; 李金环; 寿佳
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2017-05-16
Filing date: 2017-05-16
Publication date: 2021-02-23
Anticipated expiration: 2037-05-16
Also published as: CN106939303A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a Cas9 nuclease and application thereof. The Cas9 nuclease (Cas9-R919P) has Cas9 nuclease activity and is suitable for a CRISPR/Cas9 system, and the Cas9 nuclease (Cas9-R919P) is obtained by mutating 919-site arginine of a wild-type Cas9 nuclease into proline. The Cas9 nuclease (Cas9-R919P) is adopted to cut the double strand of DNA to generate a protruding broken end, bases complementary to the protruding broken end can be added in a filling-in connection mode, and accurate editing of a specific position of a genome DNA fragment can be achieved.

Description

Cas9 nuclease R919P and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Cas9 nuclease R919P and application thereof.

Background

Since the completion of the Human Genome Project (Human Genome Project) and DNA element Encyclopedia (Encyclopedia of DNA Elements) projects, scientists have analyzed and identified a large number of genes and DNA regulatory Elements in the Genome [1,2 ]. DNA regulatory elements that play an important role in gene expression regulation include promoters, enhancers, silencers, insulators, and the like. However, the function of most regulatory elements has not been experimentally verified and elucidated [2-8 ]. The function of genes and DNA regulatory elements can be explored by editing DNA fragments in genetics.

Early gene editing and gene functional modification was achieved by gene transposition and transgenesis [9-14 ]. Reverse genetics was applied to make specific mutations in genomes with the development of sequencing technologies [15,16 ]. In particular, gene-targeted mice that rely on homologous recombination are rapidly being used in scientific research [15,17,18 ]. In addition, inversion and duplication of DNA fragments in mice and zebrafish was applied to study specific genomic structural changes [19-24 ].

In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated nucleic acid 9(Cas9), CRISPR/Cas 9) derived from bacteria and archaea is an emerging genome editing technology [25-27], and is rapidly applied to eukaryotic genome editing due to its simple design and convenient operation. We achieved DNA fragment genetic editing (deletion, inversion and duplication) in human cell lines and mice using the CRISPR/Cas9 system [28 ]. After two-site targeted fragmentation is carried out in a genome by Cas 9and two sgRNAs, deletion, inversion (inversion), repetition, translocation and insertion (if a donor is provided) of a DNA fragment can be realized under the action of a repair system in which proteins such as CtIP participate [29-32 ]. Genetic manipulation of DNA fragment editing can be used to study the regulation of gene expression and three-dimensional genomic structure of tropocadherins and globin [28,31-33 ].

Editing of DNA fragments can now be achieved by the CRISPR/Cas9 system, but for in-depth studies of the precise function of specific DNA segments, Cas9 nuclease that effectively achieves precise genetic editing of DNA fragments has yet to be discovered.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a Cas9 nuclease and a use thereof.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in the first aspect of the invention, a Cas9 nuclease (Cas9-R919P) is provided, has Cas9 nuclease activity and is suitable for a CRISPR/Cas9 system, and the Cas9 nuclease is obtained by mutating 919 th arginine of a wild-type Cas9 nuclease into proline.

Preferably, the Cas9 nuclease (Cas9-R919P) cleaves a genomic DNA fragment of interest at a different ratio of protruding and blunt cleaved ends compared to wild-type Cas9 nuclease.

Preferably, the wild-type Cas9 nuclease is SpCas 9.

Further, the amino acid sequence of the wild-type Cas9 nuclease is shown in SEQ ID No. 7.

Preferably, the Cas9 nuclease (Cas9-R919P) contains an amino acid sequence shown as SEQ ID No. 9.

Preferably, the amino acid sequence of the Cas9 nuclease (Cas9-R919P) is shown as SEQ ID NO. 9.

In a second aspect of the invention, a polynucleotide is provided that encodes the Cas9 nuclease (Cas 9-R919P).

In a third aspect of the present invention, there is provided an expression vector comprising the aforementioned polynucleotide.

In a fourth aspect of the present invention, there is provided a host cell transformed with the aforementioned expression vector.

In a fifth aspect of the invention, there is provided a method for preparing the Cas9 nuclease (Cas9-R919P), comprising the steps of: constructing an expression vector containing a Cas9 nuclease (Cas9-R919P) encoding polynucleotide, then transforming the expression vector into a host cell for inducing expression, and separating the Cas9 nuclease (Cas9-R919P) from an expression product.

In a sixth aspect of the invention, there is provided a use of the foregoing Cas9 nuclease (Cas9-R919P), or a coding polynucleotide thereof, or an expression vector containing the coding polynucleotide for genomic DNA fragment editing or for preparing a genomic DNA fragment editing tool.

Preferably, the editing comprises single-site editing and multi-site editing. The editing sites of the multi-site editing are two or more.

Preferably, the editing means comprises mutation, deletion, inversion or inversion, duplication, translocation or insertion.

In the seventh aspect of the invention, a genomic DNA fragment editing tool is provided, and the genomic DNA fragment editing tool is a CRISPR/Cas9 system, and the CRISPR/Cas9 system comprises the Cas9 nuclease (Cas9-R919P) or a coding polynucleotide thereof or an expression vector containing the coding polynucleotide.

Preferably, the CRISPR/Cas9 system includes the aforementioned Cas9-R919P and one or more sgrnas for a DNA fragment of interest. The plurality means two or more.

In the eighth aspect of the invention, a method for editing a genomic DNA fragment is provided, wherein the genomic DNA fragment to be edited is edited by using the Cas9 nuclease (Cas9-R919P) and one or more sgrnas matched with the Cas9 nuclease by using the CRISPR/Cas9 system.

Preferably, an expression vector containing the polynucleotide encoding the Cas9 nuclease (Cas9-R919P) and one or more sgrnas matched with the expression vector are transferred into a cell, and the genomic DNA fragment to be edited is edited.

In the ninth aspect of the invention, a method for editing a genome DNA fragment at a single site is provided, wherein a CRISPR/Cas9 system is utilized, the Cas9 nuclease (Cas9-R919P) as claimed in claim 1 is adopted to cut a DNA double strand to generate a protruding break end, and bases complementary to the protruding break end are added in a filling-in connection manner through a cell self-repair system.

The single-site editing method of the genome DNA fragment can change the characteristics of base mutation during single-site editing.

Compared with the prior art, the invention has the following beneficial effects:

the Cas9 nuclease (Cas9-R919P) is applicable to a CRISPR/Cas9 system, the Cas9 nuclease (Cas9-R919P) contains an amino acid sequence shown as SEQ ID NO.9, and compared with a wild-type Cas9 nuclease, the Cas9 nuclease (Cas9-R919P) has different proportions of a protruding broken end and a blunt broken end generated when a target genomic DNA fragment is cut compared with the wild-type Cas9 nuclease. The Cas9 nuclease (Cas9-R919P) is adopted to cut the DNA double strand to generate a protruding fracture end, bases complementary to the protruding fracture end can be added in a filling connection mode through a cell self-repair system, and accurate editing of adding specific bases to specific positions of genome DNA fragments can be achieved.

Drawings

FIG. 1A: cas9 cleaves DNA double strands under the mediation of two sgRNAs to generate four broken ends that under the action of the cell repair system generate DNA fragment deletions, inversions and repeats.

FIG. 1B: DNA fragment deletion, inversion and duplication for HS51 site.

FIG. 1C: there is an addition of a "G" at the DNA fragment deletion junction.

FIG. 1D: there is an addition of a "T" at the DNA fragment repeat junction.

FIG. 1E: the addition of "A", "G" and "AG" was present at the reverse linker downstream of the DNA fragment.

FIG. 1F: the Cas9 cleavage pattern ratio features for the sgRNAs of these two specific sequences.

FIG. 2A: cas9 nuclease structure scheme.

FIG. 2B: schematic representation of two sgRNAs for DNA fragment editing at β -globin RE2 site.

FIG. 2C: the proportion of various cutting ends generated by each Cas9 nuclease when cutting the genomic DNA fragment under the mediation of sgRNA1 is counted by detecting the connection condition of the DNA fragment repeated joints.

FIG. 2D: the cleavage of the DNA fragment of interest was performed against the upstream sgRNA1, Cas 9and Cas9 mutants.

FIG. 2E: the proportion of various cutting ends generated by each Cas9 nuclease when cutting the genomic DNA fragment under the mediation of sgRNA2 is counted by detecting the connection condition of the DNA fragment deletion joints.

FIG. 2F: cleavage of the DNA fragment of interest by the downstream sgRNA2, Cas9, and Cas9 mutants.

FIG. 2G: cas 9and Cas9 mutants had actual and predicted ratios of base additions at the linker on the reverse side of the DNA fragment.

FIG. 3A: cleavage of the DNA fragment of interest at the STM site against the upstream sgRNA1, Cas9, and Cas9 mutants.

FIG. 3B: cleavage of the DNA fragment of interest at the STM site against downstream sgRNA2, Cas9, and Cas9 mutants.

Detailed Description

One, Cas9 nuclease

The Cas9 nuclease (Cas9-R919P) has Cas9 nuclease activity and is suitable for a CRISPR/Cas9 system, and the Cas9 nuclease is obtained by mutating 919 arginine of a wild-type Cas9 nuclease into proline. The Cas9 nuclease (Cas9-R919P) cleaves a genomic DNA fragment of interest at a different ratio of protruding and blunt cleaved ends compared to wild-type Cas9 nuclease. Further, the wild-type Cas9 nuclease is SpCas 9. Further, the amino acid sequence of the wild-type Cas9 nuclease is shown in SEQ ID No. 7.

Further, the Cas9 nuclease (Cas9- -R919P) contains an amino acid sequence shown as SEQ ID NO. 9. In some embodiments of the invention, the amino acid sequence of Cas9- -R919P is exemplified as set forth in SEQ ID No. 9.

II, polynucleotide for encoding Cas9 nuclease

The polynucleotide encoding the Cas9 nuclease (Cas9- -R919P) can be in DNA form or RNA form. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded.

Polynucleotides encoding the Cas9 nuclease (Cas9 — R919P) can be prepared by any suitable technique well known to those of skill in the art. Such techniques are described generally in the art, e.g., in the molecular cloning guidelines (J. SammBruk et al, scientific Press, 1995). Including but not limited to recombinant DNA techniques, chemical synthesis, and the like.

In some embodiments of the invention, the polynucleotide encoding the Cas9 nuclease (Cas9- -R919P) is exemplified as set forth in SEQ ID No. 10.

Expression vector

The expression vector contains a polynucleotide encoding the Cas9 nuclease (Cas 9-R919P). Methods well known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA techniques, DNA synthesis techniques and the like. The DNA of Cas9 nuclease (Cas9-R919P) can be operably linked to a multiple cloning site in a vector to direct mRNA synthesis and thus protein expression.

Fourth, host cell

The host cell is transformed with an expression vector expressing the Cas9 nuclease (Cas 9-R919P). The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are Escherichia coli, Streptomyces; salmonella typhimurium, listeria; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS.293 cells, or Bowes melanoma cells.

Fifth, method for preparing Cas9 nuclease (Cas9-R919P)

A method of making the foregoing Cas9 nuclease (Cas9-R919P), comprising the steps of: constructing an expression vector containing a polynucleotide sequence coded by a Cas9 nuclease (Cas9-R919P), then transforming the expression vector into a host cell for inducing expression, and separating the Cas9 nuclease (Cas9-R919P) from an expression product.

One skilled in the art can select suitable expression vectors and host cells based on the nature of Cas9 nuclease (Cas 9-R919P).

Use of Cas9 nuclease (Cas9-R919P) or encoding polynucleotide thereof or expression vector containing encoding polynucleotide

The Cas9 nuclease (Cas9-R919P) or encoding polynucleotide thereof or expression vector containing the encoding polynucleotide can be used for genome DNA fragment editing or for preparing genome DNA fragment editing tools.

Further, the editing includes single-site editing and multi-site editing. The editing sites of the multi-site editing are two or more. The editing modes comprise mutation, deletion, inversion or inversion, repetition, translocation or insertion.

Seven, genome DNA fragment editing tool

The genomic DNA fragment editing tool of the invention can be a CRISPR/Cas9 system comprising the aforementioned Cas9 nuclease (Cas9-R919P) or its encoding polynucleotide or an expression vector containing said encoding polynucleotide. Further, the CRISPR/Cas9 system also includes one or more sgrnas for a DNA fragment of interest. The sgRNA is designed aiming at a target DNA fragment, under the mediation of the sgRNA, Cas9-R919P can cut a DNA double strand at the upstream of a PAM (promoter adjacent motif) site to form DNA double strand break, and the precise editing of the DNA fragment is completed through a cell self-repair system. The sgRNA for the gene of interest may be one or two or more. When the sgRNA is one, single-site editing of a target DNA fragment can be achieved, and when the sgRNA is two or more, multi-site editing of the target DNA fragment can be achieved.

Eight, genome DNA fragment editing method

The genomic DNA fragment editing method adopts the Cas9 nuclease (Cas9-R919P) and one or more sgRNAs matched with the Cas9 nuclease to edit a genomic DNA fragment to be edited by using a CRISPR/Cas9 system. The editing includes single-site editing and multi-site editing. The editing sites of the multi-site editing are two or more. When the sgRNA is one, single-site editing of a target DNA fragment can be achieved, and when the sgRNA is two or more, multi-site editing of the target DNA fragment can be achieved. Further, an expression vector of the Cas9 nuclease (Cas9-R919P) -encoding polynucleotide and one or more sgrnas matched with the expression vector can be transferred into a cell, and the genomic DNA fragment to be edited can be edited.

Nine, genome DNA fragment single site editing method

By using a CRISPR/Cas9 system, the Cas9 nuclease (Cas9-R919P) provided by the invention is adopted to cut a DNA double strand to generate an outstanding fracture end, and bases complementary to the outstanding fracture end are added in a filling-in connection manner, so that single-site editing of a genome DNA fragment can be realized. The single-site editing method of the genome DNA fragment can change the characteristics of base mutation during single-site editing.

The filling connection means that: the overhanging cleaved ends are ligated after base-complementary pairing has been added to blunt ends complementary to the overhanging ends.

As exemplified in some embodiments of the invention, upon cleavage of a genomic DNA fragment (β -globin RE2 site) by Cas9 nuclease R919P mediated by sgRNA1, the resulting overhanging cleaved end U4, under the action of a cellular repair system, will first fill in the blunt end with base G complementary to overhanging end C by base complementary pairing and then be ligated to a linker.

When the Cas9 nuclease R919P cuts a genome DNA fragment (beta-globin RE2 site) under the mediation of sgRNA2, the generated protruding broken tail end D4 is added with a base T which is complementary with the protruding tail end A through base complementary pairing to fill a blunt end and then connected with a connecting joint under the action of a cell repair system.

Description of the drawings:

in the present invention, Cas9 can be used as an abbreviation for Cas9 nuclease, which means the same as Cas9 nuclease. In the invention, the mutants of Cas9-R919P, R919P and R919P are used interchangeably, and mean that the mutants are Cas9 nuclease named as R919P.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 investigation of ligation of DNA fragment editing linkers to discover a novel mechanism for Cas9 cleavage

sgRNAs plasmid for HS51 site was constructed for HS51 site:

(1) purchasing primers

Forward and reverse deoxyoligonucleotides having 5' overhang ends "ACCG" and "AAAC" that can complementarily pair against the HS51 site and sgRNAs targeting sequence, respectively, were purchased from shanghai sony biotechnology ltd;

sgRNAs targeting sequence to HS51 site described above:

HS51 RE1sgRNA1：GCCACACATCCAAGGCTGAC(SEQ ID NO.1)

HS51 RE1sgRNA2：GAGATTTGGGGCGTCAGGAAG(SEQ ID NO.2)

(2) obtaining complementary paired double-stranded DNA with overhang end

1) By ddH₂O dissolving the deoxyoligonucleotide to 100 mu M and diluting to 20 mu M;

2) the positive and negative deoxyoligonucleotide is added into the following reaction system:

reaction conditions are as follows: water bath at 95 deg.C for 5min, opening the cover of the water bath kettle, cooling to about 60 deg.C, covering the cover, and cooling to room temperature.

(3) Enzyme digestion pGL3-U6-sgRNA-PGK-Puro vector

1) The vector plasmid was digested with BsaI restriction enzyme in the following reaction scheme:

reaction conditions are as follows: 1.5 hours at 37 ℃;

2) gel recovery purification of the DNA digestion fragment and purification according to the gel recovery kit (Axygen).

(4) Ligation of the digested vector to double-stranded DNA with a overhang

The linking system is as follows:

reaction conditions are as follows: reacting at 37 ℃ for 1.5 hours at room temperature;

(5) conversion of ligation products

The ligation products were competent-transformed with Stbl3, cultured overnight on LB plates containing ampicillin (Amp, 100mg/L),

(6) picking monoclonal sequencing

1) Picking single colony from ampicillin (LB) plate, and culturing LB (Amp, 100mg/L) liquid overnight;

2) extracting plasmids according to the specification of a plasmid miniprep kit (Axygen);

3) the extracted plasmid was sequenced by Shanghai Sangni Biotech Co., Ltd.

(7) Successfully sequenced plasmid was extracted

1) Successfully sequenced plasmids were re-transformed with Stbl3 competence and cultured overnight on LB plates containing Amp (100 mg/L);

2) picking a single colony in 2ml of LB (Amp, 100mg/L) liquid culture medium to culture for 8 hours in the morning, and then transferring the colony to 200ml of LB (Amp, 100mg/L) liquid culture medium to culture overnight;

3) the bacteria were harvested and the plasmids were extracted according to the plasmid extraction kit (Qiagen).

2. Preparation of humanized Cas9 plasmid

1) The humanized Cas9 plasmid was obtained from the laboratory of the university of beijing chai jianzhong;

2) competent retransformation with Stbl3, overnight incubation on LB plates (Amp, 100 mg/L);

3) in the morning, a single colony was picked and cultured in 2ml of LB (Amp, 100mg/L) liquid medium for 8 hours, and then transferred to 200ml of LB (Amp, 100mg/L) liquid medium for overnight culture, and plasmid extraction was performed.

3. Cell transfection with Lipofectamine 2000

1) HEK293T cells were cultured in flasks at 37 ℃ with 5% CO₂And (5) culturing in a cell culture box until the cells grow to 80-90% of the culture bottle.

2) The grown cells were plated in 12-well plates with DMEM complete antibody-free medium (10% fetal bovine serum, no penicillin double antibody) and cultured overnight.

3) When the cells in the 12-well plate were 80-90% long, the prepared humanized Cas9 plasmid (800ng) and sgRNAs plasmid for HS51 site (600 ng each) were transfected with Lipofectamine 2000 in duplicate for each sample.

4) Two days after transfection, cells were collected and extracted with a genome extraction kit (

Genomic DNA purification kit, Promega).

4. Preparation of high throughput sequencing libraries

Primers were designed approximately 30bp upstream of the precise ligation site where deletion, inversion and repeat ligation of the DNA fragment was expected, then the 5' end of the primer was ligated with Illumina sequencing linker with barcode, and the downstream primer could be designed at a position away from the splicing site and ligated with Illumina sequencing linker, PCR amplification was performed, then purification was performed using Roche PCR purification kit (Product No.:11732676001), the DNA Product was dissolved in 10mM Tris-HCL buffer (pH 8.5), and the equal amount was mixed to form a library for high throughput sequencing.

High-throughput primers:

Hiseq-hHs51-aF:

ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAAGGAGATCCGTGTCGTC(SEQ ID NO.3)

Hiseq-hs51-aRa：

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGATGTTGTGGAAGGCGAGCAG(SEQ ID NO.4)

Hiseq-hs51-bFa:

CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTTACATGACAGCTTCCGGTAG(SEQ ID NO.5)

Hiseq-hHs51-bR:

CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTGGCTAACAACATAGTGCTTC(SEQ ID NO.6)。

5. high throughput sequencing data processing

After the high-throughput sequencing was completed, the sequencing results of the samples were separated from the library by barcode using the Linux program, stored in respective folders, and subjected to BWA-MEM alignment, and the aligned sequences were analyzed for insertion and deletion mutations of DNA fragments by the Varscan2 program (V2.3.9), with the Varscan2 program parameters as follows:

the invention discovers a novel mechanism for Cas9 cleavage by studying the end connection condition of DNA fragment editing.

As shown in fig. 1A, when editing a genomic DNA fragment using a sgRNA combination formed by two sgRNAs and Cas9 nuclease, Cas9 nuclease cleaves a genomic DNA double strand under the mediation of two sgRNAs to generate four break ends (DSBs) that generate DNA fragment editing such as DNA fragment deletion, inversion and duplication under the action of a cell repair system (e.g., MRN/CtIP).

As shown in fig. 1B, we edited the genomic DNA fragment HS51 RE1(HS51 site) with sgRNA1 and sgRNA2 forming a sgRNA combination and Cas9 nuclease. Then, we detected the deletion, inversion and duplication of DNA fragments, and then detected the deletion, inversion and duplication of the ligated adaptors by high throughput sequencing, except for the perfect ligation (Joined ligation) that is consistent with the expectation, there was a certain proportion of base additions (insertition) at the DNA fragment deleted ligated adaptor, the inverted downstream ligated adaptor and the duplication ligated adaptor.

As shown in FIG. 1C, when the DNA fragments were tested by high throughput sequencing to delete the ligated adaptors, the proportion of the perfect ligation (Joined Precisely) that was expected was 79.23%, and the proportion of the "G" base added at the deleted adaptors (insert, compared to the expected perfect ligation) was 11.13%.

Compared with the expected precise connection, the base of the 'G' added at the joint of the deleted DNA fragment is supposed to be a base near the PAM upstream 3bp (specifically 4bp upstream of the PAM) derived from the template DNA (HS51 RE1, HS51 site). Therefore, it is presumed that when the Cas9 nuclease cleaves a DNA strand complementary to the sgRNA, cleavage is performed 3bp upstream of the PAM; and when the Cas9 nuclease cuts a DNA strand which is not complementary with the sgRNA, the DNA strand can be cut at 4bp which is more distant from 3bp upstream of the PAM. Based on the addition of a "G" base at the DNA fragment deletion junction linker (compared to the expected precise ligation), Cas9 nuclease was presumed to have blunt end cleavage and protruding end cleavage when cleaving genomic DNA fragments under sgRNA2 mediation, thereby generating different cleaved ends. When Cas9 nuclease cleaved blunt ends of the genomic DNA fragment mediated by sgRNA2, i.e., Cas9 nuclease cleaved both the DNA strand complementary to the sgRNA and the non-complementary DNA strand 3bp upstream of the PAM, a blunt cleaved end "E3" was generated. The blunt end "E3" does not result in the addition of "G" bases at the DNA fragment deletion junction when the DNA fragment deletion is generated by the cell repair system, but rather generates a perfect ligation (Joined Precisely) in accordance with the expectation. When the Cas9 nuclease cleaves overhanging ends of a genomic DNA fragment mediated by sgRNA2, i.e., the Cas9 nuclease cleaves the DNA strand complementary to the sgRNA at 3bp upstream of the PAM, and cleaves the DNA strand non-complementary to the sgRNA at 4bp upstream of the PAM, a 5' overhanging cleaved end "E4" is generated. The 5' protruding break end "E4" when used by a cellular repair system to effect deletion of a DNA fragment results in the addition of a "G" base at the junction of the DNA fragment deletion.

Thus, we consider: cleavage with Cas9 nuclease produced a blunt cleavage end, E3, of the cleaved ends, 79.23% of the exact ligation (Joined cleavage) expected to be consistent. The proportion of the protruding cleavage end E4 was 11.13% based on "G".

However, we observed a random base deletion (Small deletion) in addition to the two major categories of exact ligation (Joined Precisely) and the addition of "G" bases at the DNA fragment deletion junction consistent with expectations. It is considered that such random base deletions (Small deletions) are generated randomly at each cleaved end (blunt cleaved end E3 and protruding cleaved end E4) under the action of the cell repair system, and the base deletions (Small deletions) are generated at each cleaved end with equal probability, and the number of base deletions (Small deletions) generated at each cleaved end under the action of the cell repair system is proportional to the number of each cleaved end.

Based on the existence of the random base deletion phenomenon, the difference exists between the actual measurement ratio of each broken end obtained by sequencing and the real ratio of the broken end, and the correction reduction is needed, namely the ratio of each broken end is calculated by taking the sum of the actual measurement ratios of the broken ends as the reference, and the ratio is taken as the occupation ratio of the broken end. That is, the proportion of each cleaved end resulting from cleavage with Cas9 nuclease was normalized and the proportion of blunt cleaved ends E3 was 87.7% [ the calculation method is: 79.23% + 11.13% (79.23% + 11.13%) ]. The proportion of protruding broken ends E4 was 12.3% [ calculated method: 11.13% + 11.13% (79.23% + 11.13%) ]. That is, Cas9 nuclease cleaved genomic DNA fragments under the mediation of sgRNA2 at 87.7% blunt end and 12.3% protruding end.

As shown in FIG. 1D, in the case of DNA fragments repeatedly ligated to adaptors by high throughput sequencing, the proportion of the perfect ligation (Joined Precisely) that was consistent with the expectation was 8.96%, and the proportion of the "T" base addition (Insertion, compared to the expected perfect ligation) at the ligated adaptor was 82.92%.

Compared with the expected precise connection, the base of the 'T' added at the repeated connection joint of the DNA fragment is supposed to be a base which is derived from the vicinity of 3bp upstream of the PAM (specifically 4bp upstream of the PAM) on the template DNA (HS51 RE1, HS51 site). Therefore, it is presumed that when the Cas9 nuclease cleaves a DNA strand complementary to the sgRNA, cleavage is performed 3bp upstream of the PAM; and when the Cas9 nuclease cuts a DNA strand which is not complementary with the sgRNA, the DNA strand can be cut at 4bp which is more distant from 3bp upstream of the PAM. Based on the detection of the presence of "T" base addition at the DNA fragment repeat ligation junction (compared to the expected precise ligation), Cas9 nuclease was postulated to have blunt end cleavage and protruding end cleavage when cleaving genomic DNA fragments under sgRNA1 mediation, thereby generating different cleaved ends. When Cas9 nuclease cleaved blunt ends of the genomic DNA fragment mediated by sgRNA1, i.e., Cas9 nuclease cleaved both the DNA strand complementary to the sgRNA and the non-complementary DNA strand 3bp upstream of the PAM, a blunt cleaved end "C3" was generated. The blunt end "C3" does not result in the addition of "T" bases at the junction of repeated DNA fragments when the DNA fragments are duplicated by the cell repair system, but rather results in a perfect ligation (Joined Precisely) that is consistent with the expectation. When the Cas9 nuclease cleaves overhanging ends of a genomic DNA fragment mediated by sgRNA1, i.e., the Cas9 nuclease cleaves the DNA strand complementary to the sgRNA at 3bp upstream of the PAM, and cleaves the DNA strand non-complementary to the sgRNA at 4bp upstream of the PAM, a 5' overhanging cleaved end "C4" is generated. The 5' overhanging split-end "C4" when acted upon by a cellular repair system to generate DNA fragment repeats, results in the addition of a "T" base at the junction of the DNA fragment repeat.

Thus, we consider: cleavage with Cas9 nuclease produced a blunt cleavage end C3 proportion of 8.96% of the cleaved ends expected to correspond to the exact ligation (Joined Precisely). The proportion of the protruding cleavage terminal C4 was 82.92% based on "T".

However, we observed a random base deletion (Small deletion) in addition to the two major categories of exact ligation (Joined Precisely) that were consistent with expectations and the addition of "T" bases at the junction of repeated ligations of DNA fragments. It is considered that such random base deletions (Small deletions) are generated randomly at each cleaved end (blunt cleaved end C3 and protruding cleaved end C4) under the action of the cell repair system, and the base deletions (Small deletions) are generated at each cleaved end with equal probability, and the number of base deletions (Small deletions) generated at each cleaved end under the action of the cell repair system is proportional to the number of each cleaved end.

Based on the existence of the random base deletion phenomenon, the difference exists between the actual measurement ratio of each broken end obtained by sequencing and the real ratio of the broken end, and the correction reduction is needed, namely the ratio of each broken end is calculated by taking the sum of the actual measurement ratios of the broken ends as the reference, and the ratio is taken as the occupation ratio of the broken end. That is, the ratio of each cleaved end resulting from cleavage with Cas9 nuclease was normalized and the ratio of blunt cleaved ends C3 was 9.75% [ the calculation method is: 8.96% + 82.92% >. The proportion of protruding broken ends C4 was 90.25% [ calculated method: 82.92% ÷ (8.96% + 82.92%) ]. That is, Cas9 nuclease cleaved genomic DNA fragments under the mediation of sgRNA1 at a ratio of 9.75% blunt-end cleavage and 90.25% overhang-end cleavage.

As shown in fig. 1E, the sequences of the generated cleaved ends were predicted according to the ratios of the way in which Cas9 nuclease cleaves the genomic DNA fragments under the mediation of sgRNA1 and sgRNA2, respectively, and the base addition conditions and ratios at the junction junctions downstream of the DNA fragment inversion were further calculated.

When Cas9 nuclease cleaves overhanging ends of a genomic DNA fragment mediated by sgRNA1 to generate an overhanging cleaved end "C4", Cas9 nuclease cleaves blunt ends of a genomic DNA fragment mediated by sgRNA2 to generate a blunt cleaved end "E3", the addition of "a" base occurs at the site where the DNA fragment reverses the downstream junction under the action of the cell repair system, and the occurrence ratio is 79.14% [ calculated as: "C4" protruding ends (90.25%) x "E3" blunt ends (87.7%) 79.14% ") was found to be similar to the experimentally detected" a "base addition at the reverse downstream adaptor of the DNA fragment at 71.94%.

When Cas9 nuclease blunt-ended cleaves the genomic DNA fragment under the mediation of sgRNA1, yielding a blunt-ended "C3", Cas9 nuclease overhanging-ended cleaves the genomic DNA fragment under the mediation of sgRNA2, yielding an overhanging broken-ended "E4", then the addition of "G" bases occurs at the downstream junction of the DNA fragment inversion under the action of the cell repair system, and the ratio occurs at 1.19% [ the calculation method is: the blunt ends of "C3" (9.75%) and the protruding ends of "E4" (12.3%) were 1.19% >) which was similar to the base addition rate of "G" at the reverse downstream adaptor of the experimentally detected DNA fragment of 8.54%.

When Cas9 nuclease cleaves overhanging ends of a genomic DNA fragment mediated by sgRNA1 to generate an overhanging cleaved end "C4", Cas9 nuclease cleaves overhanging ends of a genomic DNA fragment mediated by sgRNA2 to generate an overhanging cleaved end "E4", the addition of "AG" base occurs at the position where the DNA fragment reverses the downstream junction under the action of a cell repair system, and the occurrence ratio is 11% [ the calculation method is: the "C4" protruding break end percentage (90.25%) x "E4" protruding break end percentage (12.3%) was 11% >) similar to the experimentally detected 3.66% base addition ratio of "AG" at the reverse downstream adaptor of the DNA fragment.

When Cas9 nuclease blunt-ended cleaves the genomic DNA fragment under the mediation of sgRNA1 to generate a blunt-ended "C3", Cas9 nuclease blunt-ended cleaves the genomic DNA fragment under the mediation of sgRNA2 to generate a blunt-ended "E3", the DNA fragment reverses the downstream adaptor to ligate precisely under the action of the cell repair system and occurs in a ratio of 8.55% [ the calculation method is: the blunt-cleaved end proportion of "C3" (9.75%) x "E3" (87.7%) was 8.55%), which was similar to the experimentally detected DNA fragment inversion downstream adaptor precise ligation proportion of 6.67%.

In summary, the experimental results of fig. 1E further confirm that: when the Cas9 nuclease cuts a DNA strand that is non-complementary to the sgRNA, the cleavage can be performed from 3bp upstream of the PAM to a more distant base. Cas9 nuclease cleaves genomic DNA fragments under sgRNA-mediated cleavage with blunt-end and overhanging-end cleavage, thereby generating distinct cleaved ends. These broken ends, under the action of the cellular repair system, produce either precise DNA fragment editing that is consistent with expectations (precise editing of specific bases) or gene editing that is inconsistent with expectations (random base deletions).

As shown in fig. 1F, in the sgRNA combinations, sgrnas are designed differently (target sequences are different), and Cas9 nuclease cuts genomic DNA fragments under sgRNA-mediated conditions in different proportions, resulting in different proportions of cleaved ends. Specifically, Cas9 nuclease cleaves genomic DNA fragments under the mediation of sgRNA1 in a manner that is higher in blunt-end cleavage than in overhang-end cleavage, resulting in a blunt-break end ratio that is higher in 5' -overhang-break end ratio. However, when Cas9 nuclease cleaves a genomic DNA fragment under the mediation of sgRNA2, the proportion of overhanging ends cleaved is higher than that of blunt ends cleaved, and the proportion of 5' overhanging cleaved ends produced is also higher than that of blunt cleaved ends.

Since Cas9 nuclease was found to cleave the genomic DNA fragment under sgRNA mediation with blunt end cleavage and overhang end cleavage, bases complementary to the overhang break ends can be added in a filling-in ligation manner when Cas9 nuclease cleaves the overhang end cleavage of the genomic DNA fragment under sgRNA mediation to generate the overhang break ends, thereby achieving base addition to specific locations of the genomic DNA fragment.

Example 2 mutation of SpCas9 to obtain a specific Cas9 with altered cleavage pattern to achieve precise DNA fragment editing

1. Construction of Cas9 mutant

1) The Cas9 mutant was constructed using the NEB mutation Kit (Q5 Site-Directed Mutagenesis Kit, # E0554S) and PCR amplified first as follows:

2) KLD (Kinase, Ligase & DpnI) treatment, the reaction was as follows:

reaction conditions are as follows: room temperature for 10 minutes

3) The reaction products in 2) were all used for transformation of competent bacteria Stbl3 (50. mu.l) and cultured overnight at 37 ℃ on LB plates containing ampicillin (Amp, 100 mg/L). The single clone was picked, plasmid extracted and sequenced.

The amino acid sequence of SpCas9(Cas9WT) is shown as SEQ ID NO.7, and specifically comprises the following steps:

the encoding nucleotide sequence of the SpCas9(Cas9WT) is shown as SEQ ID NO.8, and specifically comprises the following components:

as shown in fig. 2A, Cas9 nuclease, contains RuvC domain responsible for cleaving DNA strands non-complementary to sgRNA and HNH domain responsible for cleaving DNA strands complementary to sgRNA.

The Cas9 nuclease mutant claimed by the invention is named as Cas9-R919P (arginine at position 919 of SpCas9 nuclease is mutated into proline),

the amino acid sequence of Cas9-R919P is shown as SEQ ID NO.9, and specifically comprises the following components:

the coding nucleotide sequence of Cas9-R919P is shown as SEQ ID NO.10, and specifically comprises the following components:

in addition, mutants K775A, R778A, E779A, and K918P obtained by random mutagenesis of SpCas9 were used as controls, and these control mutants were different from Cas9-R919P of the present invention in sequence.

DNA fragment editing by Cas9 nuclease mutant

(1) sgRNAs of the RRM21 site (β -globin RE2) were constructed for β -globin RE2(RRM21 site).

The sgRNAs targeting sequence:

β-globin RE2 sgRNA1：ACCCAATGACCTCAGGCTGT(SEQ ID NO.11)；

β-globin RE2 sgRNA2：TCACTTGTTAGCGGCATCTG(SEQ ID NO.12)；

forward and reverse deoxyoligonucleotides having 5' overhang "ACCG" and "AAAC" that can complementarily pair against sgRNAs targeting sequence of β -globin RE2(RRM21 site) were purchased from shanghai sony biotechnology ltd.

(2) Obtaining complementary paired double-stranded DNA with overhang end

(3) Enzyme digestion pGL3-U6-sgRNA-PGK-Puro vector

reaction conditions are as follows: 1.5 hours at 37 ℃;

(4) Ligation of the digested vector to double-stranded DNA with a overhang

The linking system is as follows:

reaction conditions are as follows: reacting for 1.5 hours at room temperature;

(5) conversion of ligation products

The ligation products were competent transformed with Stbl3 and cultured overnight at 37 ℃ on LB plates containing ampicillin (Amp, 100 mg/L).

(6) Picking monoclonal sequencing

3) the extracted plasmid was sequenced by Shanghai Sangni Biotech Co., Ltd.

(7) Successfully sequenced plasmid was extracted

(8) Cell transfection with Lipofectamine 2000

1) HEK293T cells were cultured in flasks at 37 ℃ with 5% CO₂Culturing in a cell culture box, paving the grown cells in a 12-well plate by using a DMEM (DMEM) completely non-resistant culture medium when the cells grow to 80-90% of a culture bottle, and culturing overnight;

2) when the cells in the 12-well plate grow to 80-90%, the prepared Cas 9and Cas9 mutant plasmids (800ng) and sgRNAs plasmids (600 ng each) aiming at the RRM21 site were subjected to cell transfection by Lipofectamine 2000, two replicates for each sample.

3) Two days after transfection, cells were collected and extracted with a genome extraction kit (

Genomic DNA Purification kit, Promega).

(9) Preparation of high throughput sequencing libraries

Cas9 mutant primer:

Cas9-R919P-F：CTTCATCAAAcccCAGCTTGTTGAGACACG(SEQ ID NO.13)；

Cas9-R919P-R：CCTGCTTTATCCAACTCAG(SEQ ID NO.14)；

(10) high throughput sequencing data processing

and (3) carrying out PCR amplification DNA fragment deletion, inversion and repetition by using a high-throughput sequencing primer aiming at the beta-globin RE2 locus, and establishing a library for high-throughput sequencing.

High-throughput primers:

Hiseq-RRM-1F3：

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATATGGCATCCTAGCCTTAAGAAACTAG(SEQ ID NO.15)

Hiseq-RRM-1R2：

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTACGACGCAGGAGCCGTATCATG(SEQ ID NO.16)

Hiseq-RRM-3F2：

CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATAGCAATGAAATCTTGAAGGAGTG(SEQ ID NO.17)

Hiseq-RRM-3R2：

CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCACAGCCCTGCTCTATTACG(SEQ ID NO.18)。

according to the deletion of the DNA fragment and the addition of the repeated connection joint base, the cutting mode ratio of the two sgRNAs is calculated.

Referring to the method of example 1, after editing a genomic DNA fragment by using a sgRNA combination formed by two sgRNAs and Cas9 nuclease, a high-throughput sequencing technology can be used to detect DNA fragment deletion and repeated addition of a linker base, and further calculate the ratio of blunt end cleavage mode to overhang end cleavage mode when the genomic DNA fragment is cleaved by Cas9 nuclease under the mediation of each sgRNA.

Specifically, a schematic diagram of two sgRNAs of wild-type SpCas9 nuclease (Cas9WT, WT for short) (fig. 2A) and R919P editing the genomic DNA fragment β -globin RE2 site under the mediation of each sgRNA in a sgRNA combination is shown in fig. 2B.

As shown in FIG. 2C, in the case of detecting the repeated ligation of the DNA fragments to the adapters by using the high throughput sequencing technique, in addition to the precise ligation (Joined Precisely) that is expected to be matched, there is a case where "C" bases and "GC" bases are added to the ligation adapters compared to the expected precise ligation. With different Cas9 nucleases, different ratios of perfect ligation (Joined Precisely), "+ C" bases, "+ GC" bases were detected, consistent with expectations. Taking the Cas9 nuclease chosen as R919P as an example, a percent of perfect ligation (Joined Precisely) of 64.73%, a percent of "+ C" bases of 3.27%, and a percent of "+ GC" bases of 2.11% were detected, consistent with expectations.

Given that the addition of "C" bases was detected at the DNA fragment repeat junction (compared to the expected precise ligation), we speculated that the "C" bases added at the DNA fragment repeat junction were derived from bases 4bp upstream of pam (agg) on the template DNA (β -globin RE2 site). Further, it is assumed that when the Cas9 nuclease R919P cleaves a genomic DNA fragment (β -globin RE2 site) mediated by sgRNA1, cleavage of a DNA strand complementary to sgRNA cleaves 3bp upstream of PAM, whereas cleavage of a DNA strand non-complementary to sgRNA cleaves an overhanging end 4bp upstream of PAM (agg), thereby generating an overhanging cleaved end U4. Protruding the broken end U4 results in the addition of a "C" base at the junction of the DNA segment repeat when the DNA segment repeat is generated by the cellular repair system.

Similarly, given the presence of "GC" base additions detected at DNA fragment repeat ligation junctions (compared to the expected exact ligation), we speculate that the "GC" base additions at DNA fragment repeat ligation junctions are derived from bases 4bp and 5bp upstream from pam (agg) on template DNA (β -globin RE2 site). It is further assumed that R919P, the Cas9 nuclease, cleaves a genomic DNA fragment (β -globin RE2 site) mediated by sgRNA1 at 3bp upstream of PAM for strand cleavage complementary to sgRNA, and cleaves an overhanging end at 5bp upstream of PAM (agg) for strand cleavage non-complementary to sgRNA, thereby generating an overhanging cleaved end U5. Protruding the broken end U5 results in the addition of a "GC" base at the junction of the DNA fragment repeat when it is made to repeat under the action of the cellular repair system.

When the R919P Cas9 nuclease cuts a genomic DNA fragment (beta-globin RE2 site) under the mediation of sgRNA1, a DNA strand complementary to the sgRNA is cut at 3bp upstream of PAM, and a DNA strand non-complementary to the sgRNA is cut at 3bp upstream of PAM (agg), so that a blunt-cleaved end U3 is generated. The blunt-ended U3, when used to generate DNA fragment repeats by the cell repair system, does not result in the addition of bases at the junctions where the DNA fragment repeats join, but rather generates a perfect ligation (Joined ligation) in accordance with the expectation.

Thus, we consider: cleavage with Cas9 nuclease R919P produced cleaved ends with blunt cleaved end U3 in 64.73% of the proportion of the expected consistent precision ligation (Joined precision). The proportion of the protruding cleavage end U4 was 3.27% as compared with the proportion of the "C" base. The proportion of the protruding cleavage end U5 was 2.11% based on "GC".

However, we observed a random base deletion (Small deletion) in addition to the three major categories of perfect ligation (Joined precipitation), addition of "C" bases, and addition of "GC" bases, consistent with expectations. It is considered that such random base deletions (Small deletions) are generated randomly by the action of the cell repair system at each of the cleaved ends (blunt cleavage end U3/protruding cleavage end U4/protruding cleavage end U5), and the base deletions (Small deletions) are generated at each of the cleaved ends with equal probability, and the number of base deletions (Small deletions) generated by each of the cleaved ends by the cell repair system is proportional to the number of each cleaved end.

Based on the existence of the random base deletion phenomenon, the difference exists between the actual measurement ratio of each broken end obtained by sequencing and the real ratio of the broken end, and the correction reduction is needed, namely the ratio of each broken end is calculated by taking the sum of the actual measurement ratios of the broken ends as the reference, and the ratio is taken as the occupation ratio of the broken end. That is, the proportion of each cleaved end resulting from cleavage with Cas9 nuclease R919P was normalized and calculated, the proportion of blunt cleaved end U3 was 92.32% [ the calculation method is: 64.73% ÷ (64.73% + 3.27% + 2.11%). The proportion of protruding broken ends U4 was 4.66% [ calculated method: 3.27% ÷ (64.73% + 3.27% + 2.11%) ]. The proportion of protruding broken ends U5 was 3.01% [ calculated method: 2.11% ÷ (64.73% + 3.27% + 2.11%).

That is, in the cleavage manner of the genomic DNA fragment mediated by sgRNA1 by Cas9 nuclease R919P, the rate of cleavage of the blunt end of U3 was 92.32%, the rate of cleavage of the overhanging end of U4 was 4.66%, and the rate of cleavage of the overhanging end of U5 was 3.01%.

Referring to the above method, the ratio of blunt-end cleavage of U3 to X1, protruding-end cleavage of U4 to X2, and protruding-end cleavage of U5 to X3 in the manner of cleavage of a genomic DNA fragment by a wild-type Cas9 nuclease (Cas9WT, WT for short) mediated by sgRNA1 was calculated. The results are shown in FIG. 2D and in Table 2-1 below:

TABLE 2-1

It can be seen that under the mediation of sgRNA1, when compared with SpCas9 nuclease (Cas9WT), the R919P mutant Cas9 cuts a DNA strand which is non-complementary to sgRNA1, the ratio of cutting at 5bp upstream of PAM is obviously increased (U5), and the ratio of cutting at 3bp and 4bp upstream of PAM is reduced (U3, U4).

As shown in FIG. 2E, in addition to the expected correct ligation (Joined Precisely), there is a case where "T" bases, "AT" bases, "CAT" bases are added to the deleted junction compared to the expected correct ligation when the DNA fragment is detected by using a high throughput sequencing technique to delete the junction. Different Cas9 nucleases were used, and the ratios of perfect ligation (Joined predissely), "+ T" bases, "+ AT" bases, "+ CAT" bases that were detected as being consistent with the expectations were different. Taking the Cas9 nuclease chosen as R919P as an example, the percent of perfect ligation (Joined precipitation) was 13.56%, the percent of "+ T" bases was 15.31%, the percent of "+ AT" bases was 12.69%, and the percent of "+ CAT" bases was 2.50% in agreement with expectations.

Given that the addition of "T" bases was detected at the DNA fragment deletion junction (compared to the expected precise ligation), we speculated that the "T" bases added at the DNA fragment deletion junction were derived from bases 4bp upstream of pam (tgg) on the template DNA (β -globin RE2 site). Further, it is assumed that when the Cas9 nuclease R919P cleaves a genomic DNA fragment (β -globin RE2 site) mediated by sgRNA2, cleavage of a DNA strand complementary to sgRNA cleaves 3bp upstream of PAM, and cleavage of a DNA strand non-complementary to sgRNA cleaves an overhanging end 4bp upstream of PAM (tgg), thereby generating an overhanging cleaved end D4. The addition of a "T" base at the junction of the DNA fragment deletion occurs when the overhanging split end D4 produces a DNA fragment deletion under the influence of the cellular repair system.

Similarly, given that the addition of "AT" bases was detected AT the DNA fragment deletion junction (compared to the expected exact ligation), we speculated that the "AT" bases added AT the DNA fragment deletion junction were bases derived from 4bp and 5bp upstream of pam (tgg) on the template DNA (β -globin RE2 site). It is further assumed that R919P, the Cas9 nuclease, cleaves a genomic DNA fragment (β -globin RE2 site) mediated by sgRNA2, cleaves a DNA strand complementary to sgRNA at 3bp upstream of PAM, and cleaves a DNA strand non-complementary to sgRNA at 5bp upstream of PAM (tgg), thereby generating an overhanging cleaved end D5. The addition of an "AT" base AT the junction of the DNA fragment deletion results when the DNA fragment deletion is generated by the action of the cell repair system by protruding the cleaved end D5.

Similarly, in view of the fact that the addition of "CAT" base was detected at the DNA fragment deletion junction (compared with the expected precise ligation), we speculated that the "CAT" base added at the DNA fragment deletion junction was derived from the base 4bp, 5bp, 6bp upstream of PAM (TGG) on the template DNA (β -globin RE2 site). It is further assumed that R919P, the Cas9 nuclease, cleaves a genomic DNA fragment (β -globin RE2 site) mediated by sgRNA2, cleaves a DNA strand complementary to sgRNA at 3bp upstream of PAM, and cleaves a DNA strand non-complementary to sgRNA at 6bp upstream of PAM (tgg), thereby generating an overhanging cleaved end D6. The addition of "CAT" bases at the junction of the DNA fragment deletion occurs when the DNA fragment deletion is generated by the action of the cellular repair system by protruding the broken end D5.

When the R919P Cas9 nuclease cuts a genomic DNA fragment (beta-globin RE2 site) under the mediation of sgRNA2, a DNA strand complementary to the sgRNA is cut at 3bp upstream of PAM, and a DNA strand non-complementary to the sgRNA is cut at 3bp upstream of PAM (tgg), so that a blunt-cleaved end D3 is generated. When the blunt end D3 is used to generate DNA fragment deletion by the cell repair system, it does not result in the addition of bases at the junction of the DNA fragment deletion junction, but generates a perfect junction (Joined ligation) in accordance with the expectation.

Thus, we consider: cleavage with Cas9 nuclease R919P produced cleaved ends with blunt cleaved end D3 at 13.56% of the expected consistent precision ligation (Joined precision). The proportion of the overhanging cleavage end D4 to the base "T" was 15.31%. The proportion of the overhanging cleavage end D5 to the base "AT" was 12.69%. The proportion of the protruding cleavage end D6 added was 2.50% of the "CAT" base.

However, we observed that there was a random base deletion (Small deletion) in addition to the four major cases of "T" base, "+ AT" base, "+ CAT" base added to the ligation linker for precise ligation (Joined Precisely), DNA fragment deletion, and the like that were consistent with expectations. It is considered that such random base deletions (Small deletions) are generated randomly by the action of the cell repair system at each of the cleaved ends (blunt cleavage end D3/protruding cleavage end D4/protruding cleavage end D5/protruding cleavage end D6), and the base deletions (Small deletions) are generated at each of the cleaved ends with equal probability, and the number of base deletions (Small deletions) generated by each of the cleaved ends by the cell repair system is proportional to the number of each cleaved end.

Based on the existence of the random base deletion phenomenon, the difference exists between the actual measurement ratio of each broken end obtained by sequencing and the real ratio of the broken end, and the correction reduction is needed, namely the ratio of each broken end is calculated by taking the sum of the actual measurement ratios of the broken ends as the reference, and the ratio is taken as the occupation ratio of the broken end. Namely, the proportion of each broken end generated by the cutting of Cas9 nuclease R919P is standardized, and the proportion of blunt broken end D3 is 30.78 percent

The calculation method comprises the following steps: 13.56% ÷ (13.56% + 15.31% + 12.69% + 2.50%).

The proportion of the protruding breaking end D4 was 34.75%

The calculation method comprises the following steps: 15.31% ÷ (13.56% + 15.31% + 12.69% + 2.50%).

The proportion of the protruding breaking end D5 was 28.80%

The calculation method comprises the following steps: 12.69% ÷ (13.56% + 15.31% + 12.69% + 2.50%).

The proportion of the protruding breaking end D6 was 5.67%

The calculation method comprises the following steps: 2.50% ÷ (13.56% + 15.31% + 12.69% + 2.50%).

That is, in the manner in which Cas9 nuclease R919P cleaves genomic DNA fragments under the mediation of sgRNA2, the percentage of blunt-end cleavage at D3, the percentage of overhanging-end cleavage at D4, the percentage of overhanging-end cleavage at D5, and the percentage of overhanging-end cleavage at D6 were 30.78%, 34.75%, and 28.80%, respectively.

Referring to the above method, the manner of cleavage of the genomic DNA fragment by the wild-type Cas9 nuclease mediated by sgRNA2 was calculated to be that the ratio of the blunt-end cleavage of D3 to the ratio of the overhanging-end cleavage of Y1, the ratio of the overhanging-end cleavage of D4 to the ratio of the overhanging-end cleavage of Y2, the ratio of the overhanging-end cleavage of D5 to the ratio of the overhanging-end cleavage of Y3, and the ratio of the overhanging-end cleavage of D6 to Y4. The results are shown in FIG. 2F and Table 2-2:

tables 2 to 2

It can be seen that, when the R919P mutant cleaves a DNA strand of a genomic DNA fragment that is non-complementary to sgRNA2 under the mediation of sgRNA2, the rate of cleavage at 3bp upstream of PAM is significantly increased (D3) and the rate of cleavage at 4bp upstream of PAM is significantly increased (D4) compared to SpCas9 nuclease (Cas9 WT).

Referring to the method of example 1, the sequences of the generated broken ends were predicted according to the ratio of the way in which Cas9 nuclease cuts genomic DNA fragments under the mediation of sgRNA1 and sgRNA2, respectively, and the base addition condition and ratio at the junction downstream of the DNA fragment inversion were further calculated. As shown in FIG. 2G, the calculation result was similar to the experimentally detected base addition ratio. It was further demonstrated that Cas9 nuclease can cleave non-complementary DNA strands 3bp upstream of PAM to more distant bases mediated by sgRNA combinations.

In addition, the Cas9 nuclease mutant Cas9-R919P, the control mutants K775A, R778A, E779A and K918P of the invention and two sgRNAs aiming at an STM locus (beta-globin RE1) are transfected into human embryonic kidney HEK293T cells, genomic DNA is collected after 48 hours of transfection, a high-throughput sequencing primer is used for carrying out PCR amplification DNA fragment deletion, inversion and repetition, and a library is built for high-throughput sequencing. The ratio of the cleavage pattern of these mutants at the two sgRNAs was calculated based on DNA fragment deletions and repeated addition of linker bases.

sgRNAs targeting sequence to STM site (β -globin RE 1):

β-globin RE1sgRNA1：GATTGTTGTTGCCTTGGAGTG(SEQ ID NO.19)；

β-globin RE1sgRNA2：GCTGGTCCCCTGGTAACCTGG(SEQ ID NO.20)；

forward and reverse deoxyoligonucleotides:

β-globin RE1sgRNA1F：accgATTGTTGTTGCCTTGGAGTG(SEQ ID NO.21)；

β-globin RE1sgRNA1R：aaacCACTCCAAGGCAACAACAAT(SEQ ID NO.22)；

β-globin RE1sgRNA2F：accgCTGGTCCCCTGGTAACCTGG(SEQ ID NO.23)；

β-globin RE1sgRNA2R：aaacCCAGGTTACCAGGGGACCAG(SEQ ID NO.24)；

high-throughput primers:

Hiseq-hSTM-aF1:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCTTAGAGCCAGGACTAATTGC(SEQ ID NO.25)；

Hiseq-hSTM-aR2:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGGTGTAGAAATGAGCAAATAAGT(SEQ ID NO.26)；

Hiseq-hSTM-2F:

CAAGCAGAAGACGGCATACGAGATGATCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGATTGAGTTCTGTTTGTTTCATCTAC(SEQ ID NO.27)；

Hiseq-hSTM-2R:

CAAGCAGAAGACGGCATACGAGATAGTCAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCTCTGCCTGAAAGGAGTC(SEQ ID NO.28)。

as shown in fig. 3A and 3B, compared to the wild-type SpCas9 nuclease (Cas9WT for short), the control mutants K775A, R778A, E779A and K918P did not significantly change the way the genomic DNA fragments were cleaved under the mediation of sgRNA1 and sgRNA 2; compared with a wild SpCas9 nuclease (Cas9WT for short), the Cas9 nuclease mutant Cas9-R919P has obviously changed the way of cutting a genome DNA fragment under the mediation of sgRNA1 and sgRNA 2.

In conclusion, the Cas9 nuclease (Cas9-R919P) of the present invention has a different ratio of protruding and blunt cleaved ends when cleaving a genomic DNA fragment of interest compared to the wild-type Cas9 nuclease. By adopting the Cas9 nuclease (Cas9-R919P) disclosed by the invention, the specific position of a target genome DNA fragment can be cut, the protruding broken end can be generated, the base complementary with the protruding broken end can be added in a filling-in connection mode, and further, the accurate DNA fragment editing of the specific position can be realized.

The references of the present application are as follows:

1.Stamatoyannopoulos,JA.(2012).What does our genome encodeGenome Res,22:1602-1611.

2.The ENCODE Project Consortium.(2012).An integrated encyclopedia of DNA elements in the human genome.Nature,489:57-74.

3.Banerji,J,L Olson,and W Schaffner.(1983).A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes.Cell,33:729-740.

4.Zhang,T,P Haws,and Q Wu.(2004).Multiple variable first exons:a mechanism for cell-and tissue-specific gene regulation.Genome Res,14:79-89.

5.Neph,S,et al.(2012).An expansive human regulatory lexicon encoded in transcription factor footprints.Nature,489:83-90.

6.Shen,Y,et al.(2012).A map of the cis-regulatory sequences in the mouse genome.Nature,488:116-120.

7.Thurman,RE,et al.(2012).The accessible chromatin landscape of the human genome.Nature,489:75-82.

8.de Laat,W and D Duboule.(2013).Topology of mammalian developmental enhancers and their regulatory landscapes.Nature,502:499-506.

9.McClintock,B.(1950).The origin and behavior of mutable loci in maize.Proc Natl Acad Sci U S A,36:344-355.

10.McClintock,B.(1984).The significance of responses of the genome to challenge.Science,226:792-801.

11.Brinster,RL,et al.(1981).Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs.Cell,27:223-231.

12.Harbers,K,D Jahner,and R Jaenisch.(1981).Microinjection of cloned retroviral genomes into mouse zygotes:integration and expression in the animal.Nature,293:540-542.

13.Gordon,JW,et al.(1980).Genetic transformation of mouse embryos by microinjection of purified DNA.Proc Natl Acad Sci U S A,77:7380-7384.

14.Palmiter,RD,et al.(1982).Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes.Nature,300:611-615.

15.Capecchi,MR.(2005).Gene targeting in mice:functional analysis of the mammalian genome for the twenty-first century.Nat Rev Genet,6:507-512.

16.Carroll,D.(2014).Genome engineering with targetable nucleases.Annu Rev Biochem,83:409-439.

17.Smithies,O,et al.(1985).Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination.Nature,317:230-234.

18.Thomas,KR and MR Capecchi.(1986).Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene.Nature,324:34-38.

19.Zheng,B,et al.(2000).Engineering mouse chromosomes with Cre-loxP:range,efficiency,and somatic applications.Mol Cell Biol,20:648-655.

20.Wu,S,et al.(2007).Toward simpler and faster genome-wide mutagenesis in mice.Nat Genet,39:922-930.21.Gupta,A,et al.(2013).Targeted chromosomal deletions and inversions in zebrafish.Genome Res,23:1008-1017.

22.Xiao,A,et al.(2013).Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.Nucleic Acids Res,41:e141.

23.Kraft,K,et al.(2015).Deletions,Inversions,Duplications:Engineering of Structural Variants using CRISPR/Cas in Mice.Cell Rep,10:833-839.

24.Wu,S,et al.(2008).A protocol for constructing gene targeting vectors:generating knockout mice for the cadherin family and beyond.Nature Protocol,3:1056-1076.

25.Jinek,M,et al.(2012).A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,337:816-821.

26.Cong,L,et al.(2013).Multiplex genome engineering using CRISPR/Cas systems.Science,339:819-823.

27.Mali,P,et al.(2013).RNA-guided human genome engineering via Cas9.Science,339:823-826.

28.Li,J,et al.(2015).Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.J Mol Cell Biol,7:284-298.

29.Sartori,AA,et al.(2007).Human CtIP promotes DNA end resection.Nature,450:509-514.

30.Anand,R,et al.(2016).Phosphorylated CtIP Functions as a Co-factor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection.Mol Cell,64:940-950.

31.Li,J,J Shou,and Q Wu.(2015).DNA fragment editing of genomes by CRISPR/Cas9.Hereditas,37:992-1002.

32.Huang,H and Q Wu.(2016).CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes.J Genet Genomics,43:273-288.

33.Guo,Y,et al.(2015).CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function.Cell,162:900-910.

while the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shanghai university of transportation

<120> Cas9 nuclease R919P and application thereof

<130> 171288

<160> 28

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial

<220>

<223> HS51 RE1sgRNA1

<400> 1

gccacacatc caaggctgac 20

<210> 2

<211> 21

<212> DNA

<213> Artificial

<220>

<223> HS51 RE1sgRNA2

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gagatttggg gcgtcaggaa g 21

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<212> DNA

<213> Artificial

<220>

<223> Hiseq-hHs51-aF

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atgatacggc gaccaccgag atctacactc tttccctaca cgacgctctt ccgatctgca 60

aggagatccg tgtcgtc 77

<210> 4

<211> 82

<212> DNA

<213> Artificial

<220>

<223> Hiseq-hs51-aRa

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aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctaa 60

ggatgttgtg gaaggcgagc ag 82

<210> 5

<211> 87

<212> DNA

<213> Artificial

<220>

<223> Hiseq-hs51-bFa

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caagcagaag acggcatacg agatggacgg gtgactggag ttcagacgtg tgctcttccg 60

atctctttac atgacagctt ccggtag 87

<210> 6

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<212> DNA

<213> Artificial

<220>

<223> Hiseq-hHs51-bR

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caagcagaag acggcatacg agatttgact gtgactggag ttcagacgtg tgctcttccg 60

atcttttttg gctaacaaca tagtgcttc 89

<210> 7

<211> 1401

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<220>

<223> SpCas9

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Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala

1 5 10 15

Ala Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser

20 25 30

Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys

35 40 45

Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu

50 55 60

Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg

65 70 75 80

Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile

85 90 95

Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp

100 105 110

Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys

115 120 125

Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala

130 135 140

Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val

145 150 155 160

Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala

165 170 175

His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn

180 185 190

Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr

195 200 205

Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp

210 215 220

Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu

225 230 235 240

Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly

245 250 255

Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn

260 265 270

Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr

275 280 285

Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala

290 295 300

Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser

305 310 315 320

Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala

325 330 335

Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu

340 345 350

Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe

355 360 365

Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala

370 375 380

Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met

385 390 395 400

Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu

405 410 415

Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His

420 425 430

Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro

435 440 445

Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg

450 455 460

Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala

465 470 475 480

Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu

485 490 495

Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met

500 505 510

Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His

515 520 525

Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val

530 535 540

Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu

545 550 555 560

Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val

565 570 575

Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe

580 585 590

Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu

595 600 605

Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu

610 615 620

Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu

625 630 635 640

Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr

645 650 655

Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg

660 665 670

Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg

675 680 685

Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly

690 695 700

Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr

705 710 715 720

Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser

725 730 735

Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys

740 745 750

Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met

755 760 765

Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn

770 775 780

Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg

785 790 795 800

Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His

805 810 815

Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr

820 825 830

Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn

835 840 845

Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu

850 855 860

Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn

865 870 875 880

Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met

885 890 895

Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg

900 905 910

Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu

915 920 925

Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile

930 935 940

Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr

945 950 955 960

Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys

965 970 975

Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val

980 985 990

Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala

995 1000 1005

Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser

1010 1015 1020

Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1025 1030 1035

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr

1040 1045 1050

Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr

1055 1060 1065

Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn

1070 1075 1080

Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala

1085 1090 1095

Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys

1100 1105 1110

Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu

1115 1120 1125

Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp

1130 1135 1140

Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr

1145 1150 1155

Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys

1160 1165 1170

Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg

1175 1180 1185

Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly

1190 1195 1200

Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1205 1210 1215

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1220 1225 1230

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys

1235 1240 1245

Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys

1250 1255 1260

Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln

1265 1270 1275

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe

1280 1285 1290

Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu

1295 1300 1305

Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala

1310 1315 1320

Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro

1325 1330 1335

Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr

1340 1345 1350

Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser

1355 1360 1365

Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly

1370 1375 1380

Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys

1385 1390 1395

Lys Lys Lys

1400

<210> 8

<211> 4206

<212> DNA

<213> Artificial

<220>

<223> SpCas9

<400> 8

atggccccaa agaagaagcg gaaggtcggt atccacggtg tcccagcagc catggacaag 60

aagtactcca ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac 120

gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 180

aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg 240

ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 300

gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 360

tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 420

gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 480

gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 540

tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 600

ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 660

tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 720

aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 780

ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 840

cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 900

gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 960

gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 1020

cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 1080

cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 1140

gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 1200

gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 1260

actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactc 1320

aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 1380

ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 1440

tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 1500

aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 1560

aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 1620

ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 1680

cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 1740

ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 1800

gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 1860

aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 1920

acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 1980

gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 2040

agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 2100

aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 2160

tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac 2220

atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc 2280

gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg 2340

gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg 2400

attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac 2460

acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac 2520

gtggatcagg aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc 2580

cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat 2640

agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg 2700

cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct 2760

gaacgaggtg gcctgtctga gttggataaa gcaggcttca tcaaaaggca gcttgttgag 2820

acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac 2880

gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc 2940

tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat 3000

gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag 3060

cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca 3120

aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg 3180

aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc 3240

gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc 3300

cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga 3360

ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa 3420

aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta 3480

ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg 3540

ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag 3600

gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc 3660

tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt 3720

aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa 3780

aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa 3840

cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc 3900

gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg 3960

gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc 4020

ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg 4080

gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct 4140

cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagc taagaaaaag 4200

aaataa 4206

<210> 9

<211> 1401

<212> PRT

<213> Artificial

<220>

<223> Cas9- R919P

<400> 9

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala

1 5 10 15

Ala Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser

20 25 30

Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys

35 40 45

Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu

50 55 60

Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg

65 70 75 80

Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile

85 90 95

Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp

100 105 110

Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys

115 120 125

Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala

130 135 140

Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val

145 150 155 160

Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala

165 170 175

His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn

180 185 190

Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr

195 200 205

Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp

210 215 220

Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu

225 230 235 240

Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly

245 250 255

Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn

260 265 270

Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr

275 280 285

Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala

290 295 300

Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser

305 310 315 320

Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala

325 330 335

Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu

340 345 350

Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe

355 360 365

Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala

370 375 380

Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met

385 390 395 400

Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu

405 410 415

Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His

420 425 430

Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro

435 440 445

Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg

450 455 460

Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala

465 470 475 480

Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu

485 490 495

Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met

500 505 510

Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His

515 520 525

Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val

530 535 540

Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu

545 550 555 560

Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val

565 570 575

Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe

580 585 590

Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu

595 600 605

Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu

610 615 620

Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu

625 630 635 640

Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr

645 650 655

Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg

660 665 670

Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg

675 680 685

Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly

690 695 700

Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr

705 710 715 720

Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser

725 730 735

Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys

740 745 750

Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met

755 760 765

Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn

770 775 780

Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg

785 790 795 800

Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His

805 810 815

Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr

820 825 830

Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn

835 840 845

Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu

850 855 860

Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn

865 870 875 880

Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met

885 890 895

Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg

900 905 910

Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu

915 920 925

Asp Lys Ala Gly Phe Ile Lys Pro Gln Leu Val Glu Thr Arg Gln Ile

930 935 940

Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr

945 950 955 960

Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys

965 970 975

Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val

980 985 990

Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala

995 1000 1005

Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser

1010 1015 1020

Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1025 1030 1035

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr

1040 1045 1050

Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr

1055 1060 1065

Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn

1070 1075 1080

Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala

1085 1090 1095

Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys

1100 1105 1110

Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu

1115 1120 1125

Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp

1130 1135 1140

Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr

1145 1150 1155

Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys

1160 1165 1170

Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg

1175 1180 1185

Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly

1190 1195 1200

Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1205 1210 1215

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1220 1225 1230

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys

1235 1240 1245

Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys

1250 1255 1260

Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln

1265 1270 1275

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe

1280 1285 1290

Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu

1295 1300 1305

Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala

1310 1315 1320

Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro

1325 1330 1335

Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr

1340 1345 1350

Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser

1355 1360 1365

Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly

1370 1375 1380

Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys

1385 1390 1395

Lys Lys Lys

1400

<210> 10

<211> 4206

<212> DNA

<213> Artificial

<220>

<223> Cas9- R919P

<400> 10

atggccccaa agaagaagcg gaaggtcggt atccacggtg tcccagcagc catggacaag 60

aagtactcca ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac 120

gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 180

aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg 240

ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 300

gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 360

tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 420

gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 480

gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 540

tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 600

ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 660

tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 720

aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 780

ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 840

cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 900

gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 960

gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 1020

cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 1080

cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 1140

gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 1200

gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 1260

actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactc 1320

aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 1380

ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 1440

tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 1500

aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 1560

aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 1620

ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 1680

cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 1740

ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 1800

gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 1860

aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 1920

acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 1980

gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 2040

agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 2100

aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 2160

tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac 2220

atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc 2280

gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg 2340

gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg 2400

attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac 2460

acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac 2520

gtggatcagg aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc 2580

cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat 2640

agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg 2700

cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct 2760

gaacgaggtg gcctgtctga gttggataaa gcaggcttca tcaaacccca gcttgttgag 2820

acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac 2880

gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc 2940

tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat 3000

gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag 3060

cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca 3120

aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg 3180

aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc 3240

gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc 3300

cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga 3360

ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa 3420

aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta 3480

ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg 3540

ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag 3600

gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc 3660

tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt 3720

aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa 3780

aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa 3840

cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc 3900

gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg 3960

gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc 4020

ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg 4080

gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct 4140

cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagc taagaaaaag 4200

aaataa 4206

<210> 11

<211> 20

<212> DNA

<213> Artificial

<220>

<223> β-globin RE2sgRNA1

<400> 11

acccaatgac ctcaggctgt 20

<210> 12

<211> 20

<212> DNA

<213> Artificial

<220>

<223> β-globin RE2sgRNA2

<400> 12

tcacttgtta gcggcatctg 20

<210> 13

<211> 30

<212> DNA

<213> Artificial

<220>

<223> Cas9- R919P-F

<400> 13

cttcatcaaa ccccagcttg ttgagacacg 30

<210> 14

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Cas9- R919P-R

<400> 14

cctgctttat ccaactcag 19

<210> 15

<211> 86

<212> DNA

<213> Artificial

<220>

<223> Hiseq-RRM-1F3

<400> 15

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctat 60

atggcatcct agccttaaga aactag 86

<210> 16

<211> 81

<212> DNA

<213> Artificial

<220>

<223> Hiseq-RRM-1R2

<400> 16

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctta 60

cgacgcagga gccgtatcat g 81

<210> 17

<211> 89

<212> DNA

<213> Artificial

<220>

<223> Hiseq-RRM-3F2

<400> 17

caagcagaag acggcatacg agataagcta gtgactggag ttcagacgtg tgctcttccg 60

atctatagca atgaaatctt gaaggagtg 89

<210> 18

<211> 85

<212> DNA

<213> Artificial

<220>

<223> Hiseq-RRM-3R2

<400> 18

caagcagaag acggcatacg agattcaagt gtgactggag ttcagacgtg tgctcttccg 60

atctgcacag ccctgctcta ttacg 85

<210> 19

<211> 21

<212> DNA

<213> Artificial

<220>

<223> β-globin RE1sgRNA1

<400> 19

gattgttgtt gccttggagt g 21

<210> 20

<211> 21

<212> DNA

<213> Artificial

<220>

<223> β-globin RE1sgRNA2

<400> 20

gctggtcccc tggtaacctg g 21

<210> 21

<211> 24

<212> DNA

<213> Artificial

<220>

<223> β-globin RE1sgRNA1F

<400> 21

accgattgtt gttgccttgg agtg 24

<210> 22

<211> 24

<212> DNA

<213> Artificial

<220>

<223> β-globin RE1sgRNA1R

<400> 22

aaaccactcc aaggcaacaa caat 24

<210> 23

<211> 24

<212> DNA

<213> Artificial

<220>

<223> β-globin RE1sgRNA2F

<400> 23

accgctggtc ccctggtaac ctgg 24

<210> 24

<211> 24

<212> DNA

<213> Artificial

<220>

<223> β-globin RE1sgRNA2R

<400> 24

aaacccaggt taccagggga ccag 24

<210> 25

<211> 81

<212> DNA

<213> Artificial

<220>

<223> Hiseq-hSTM-aF1

<400> 25

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60

cttagagcca ggactaattg c 81

<210> 26

<211> 83

<212> DNA

<213> Artificial

<220>

<223> Hiseq-hSTM-aR2

<400> 26

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60

ggtgtagaaa tgagcaaata agt 83

<210> 27

<211> 91

<212> DNA

<213> Artificial

<220>

<223> Hiseq-hSTM-2F

<400> 27

caagcagaag acggcatacg agatgatcgt gtgactggag ttcagacgtg tgctcttccg 60

atctagattg agttctgttt gtttcatcta c 91

<210> 28

<211> 85

<212> DNA

<213> Artificial

<220>

<223> Hiseq-hSTM-2R

<400> 28

caagcagaag acggcatacg agatagtcaa gtgactggag ttcagacgtg tgctcttccg 60

atctcagctc tgcctgaaag gagtc 85

Claims

1. Use of a Cas9 nuclease for preparing a CRISPR/Cas9 system, wherein the amino acid sequence of the Cas9 nuclease is shown as SEQ ID NO.9, the CRISPR/Cas9 system has a function of cutting a DNA double strand to generate a protruding break end, and a base function complementary to the protruding break end is added in a filling connection mode through a cell self-repair system, and the CRISPR/Cas9 system can be used for genome DNA fragment editing.

2. The use of claim 1, wherein the Cas9 nuclease cleaves genomic DNA fragments of interest at a different ratio of protruding to blunt cleaved ends compared to wild-type Cas9 nuclease.

3. The use of claim 2, wherein the wild-type Cas9 nuclease is SpCas 9.

4. The use according to claim 3, wherein the amino acid sequence of the wild-type Cas9 nuclease is as set forth in SEQ ID No. 7.

5. Use according to claim 1, wherein the editing comprises single-site editing or multi-site editing, the number of editing sites of the multi-site editing being two or more.

6. The use according to claim 5, wherein the means of multi-site editing comprises deletion, inversion, duplication, translocation or insertion of DNA fragments of different lengths.

7. A genome DNA fragment single-site editing method, which utilizes a CRISPR/Cas9 system, cuts a DNA double strand by using the Cas9 nuclease as claimed in any one of claims 1-5 to generate a protruding fracture end, and adds bases complementary to the protruding fracture end in a filling-in connection manner through a cell self-repair system to realize the insertion of specific bases.

8. The method of editing a single site of a genomic DNA fragment according to claim 7, wherein the method can change the characteristics of the base mutation in the single site editing.