CN110551763A - CRISPR/SlutCas9 gene editing system and application thereof - Google Patents

Abstract

The invention belongs to the technical field of gene editing, and particularly relates to a CRISPR/SlutCas9 gene editing system and application thereof, wherein the gene editing system is a complex formed by SlutCas9 protein and sgRNA, can accurately target a DNA sequence and generate cutting to enable the DNA to generate double-strand break damage, the gene editing is performed in cells or in vitro, SlutCas9 protein is small and is 1054 amino acids, an identified PAM sequence is simple, the SlutCas9 protein has an amino acid sequence shown by SEQ ID NO. 1, and the sgRNA has a nucleotide sequence shown by SEQ ID NO. 2.

Description

CRISPR/SlutCas9 gene editing system and application thereof

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to a CRISPR/SlutCas9 system capable of performing gene editing in cells and related application thereof.

background

CRISPR/Cas9 is an acquired immune system that bacteria and archaea have evolved to protect against foreign virus or plasmid invasion. In the CRISPR/Cas9 system, after a complex is formed by crRNA (CRISPR-derived RNA), tracrRNA (trans-activating RNA) and Cas9 protein, a pam (promoter adjacentmotif) sequence for identifying a target site can form a complementary structure with a target DNA sequence, and Cas9 protein plays a role in cutting DNA, so that the DNA is subjected to breaking damage. Wherein, tracrRNA and crRNA can be fused into single-stranded guide RNA (sgRNA) through a connecting sequence. When DNA breaks and damages, two major DNA damage repair mechanisms within the cell are responsible for repair: non-homologous end-joining (NHEJ) and Homologous Recombination (HR). Deletion or insertion of a base can be caused as a result of NHEJ repair, and gene knockout can be carried out; in the case of providing a homologous template, site-directed insertion of genes and precise base substitution can be performed using HR repair.

Besides basic scientific research, the CRISPR/Cas9 also has wide clinical application prospect. When the CRISPR/Cas9 system is used for gene therapy, Cas9 and sgRNA need to be introduced into a body. The most effective delivery vector for gene therapy is AAV. However, AAV virus-packaged DNA typically does not exceed 4.5 kb. SpCas9 has been widely used because of its simple PAM sequence (recognition of NGG) and high activity. However, the SpCas9 protein has 1367 amino acids, and the sgRNA and the promoter cannot be effectively packaged into the AAV virus, so that the clinical application of the protein is limited. To overcome this problem, several small Cas9 were invented, including SaCas9(PAM sequence NNGRRT); st1Cas9(PAM sequence NNAGAW); NmCas9(PAM sequence NNNNGATT); nme2Cas9(PAM sequence NNNNCC); cjCas9(PAM sequence is NNRYAC), but these Cas9 or PAM sequences are complex (few DNA sequences can be targeted in genome), or editing efficiency is low, and wide application is difficult. The search for a small Cas9 protein, PAM sequence simple CRISPR/Cas9 system is hopeful to solve the above problems.

disclosure of Invention

In view of the above problems, the present invention aims to provide a novel gene editing system of CRISPR/Cas9 with high editing activity, small Cas9 protein and simple PAM sequence, and applications thereof.

The CRISPR/Cas9 gene editing system provided by the invention is a complex formed by a SlutCas9 protein and a sgRNA, and is marked as a CRISPR/SlutCas9 gene editing system (namely, the SlutCas9 protein which realizes gene editing under the combined action with single guide RNA (sgRNA)); the DNA sequence can be precisely targeted, and the cutting is generated, so that the DNA is subjected to double-strand break damage; the gene editing is gene editing in a cell or in vitro; the SlutCas9 protein is small and 1054 amino acids, the identified PAM sequence is simple (NNGR), the SlutCas9 protein has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence which is at least 80% identical to the amino acid sequence shown in SEQ ID NO. 1; the sgRNA has a nucleotide sequence shown in SEQ ID NO. 2, or a sgRNA sequence modified based on SEQ ID NO. 2.

In the present invention, the cells include eukaryotic cells and prokaryotic cells; the eukaryotic cells include mammalian cells and plant cells.

In the present invention, the mammalian cells include Chinese hamster ovary cells, baby hamster kidney cells, mouse Sertoli cells, mouse mammary tumor cells, buffalo rat liver cells, rat liver tumor cells, monkey kidney CVI line transformed by SV40, monkey kidney cells, canine kidney cells, human cervical cancer cells, human lung cells, human liver cells, HIH/3T3 cells, human U2-OS osteosarcoma cells, human A549 cells, human K562 cells, human HEK293T cells, human HCT116 cells, or human MCF-7 cells or TRI cells.

In the invention, the CRISPR/Cas9 system is Staphyloccocus lutraeCas9(SlutCas9) protein, and the CRISPR/Cas9 system and single guide RNA (sgRNA) act together to realize gene editing.

In the invention, the SlutCas9 protein belongs to the genus otter Staphylococcus (Staphylococcus lutee), and the retrieval number of Uniprot of the SlutCas9 protein is A0A1W6BMI 2.

In the invention, the SlutCas9 protein comprises a SlutCas9 protein which has no cleavage activity or only single-strand cleavage activity or double-strand cleavage activity.

In the invention, the precise positioning DNA sequence comprises a sequence of 20bp or 21bp at the 5' end of the sgRNA and a target DNA sequence which can form a base complementary pairing structure.

In the invention, the accurate positioning target DNA sequence comprises a PAM sequence on the target DNA sequence recognized by the SlutCas9 protein and the sgRNA complex.

In the invention, the PAM sequence is NNGR, and the target DNA sequence is:

NNNNNNNNNNNNNNNNNNNNNNNGR(SEQ ID NO:3)。

in the invention, the SlutCas9 protein and sgRNA compound can accurately target DNA sequences, namely the SlutCas9 protein and sgRNA compound can recognize and bind specific DNA sequences, or other proteins fused with the SlutCas9 protein or proteins specifically recognizing sgRNA are brought to the position of the target DNA.

in the invention, the slotcas 9 protein and sgRNA complex or other protein fused with slotcas 9 protein or protein specifically recognizing sgRNA can modify and regulate a targeted DNA region, and the modification and regulation includes but is not limited to regulation of gene transcription level, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single-base converter or chromatin imaging tracking.

In the present invention, the single base converter includes, but is not limited to, conversion between bases adenine to guanine, or cytosine to thymine, or cytosine to uracil, or other bases.

The gene editing system provided by the invention has high editing activity and has obvious advantages compared with the prior Cas 9.

The editing efficiency of the CRISPR/SlutCas9 system is detected by the techniques of gene synthesis, molecular cloning, cell transfection, PCR product deep sequencing, bioinformatics analysis and the like.

The CRISPR/SlutCas9 gene editing system provided by the invention can carry out gene editing in cells, and comprises the steps of identifying and positioning target DNA through a compound of a SlutCas9 protein and sgRNA, and editing the DNA; and finally, detecting the editing efficiency. The method comprises the following specific steps:

(1) Synthesizing a humanized SlutCas9 gene sequence; and cloning to an expression vector to obtain pAAV2_ SlutCas9_ ITR;

(2) Synthesizing oligonucleotide single-stranded DNA (deoxyribonucleic acid) corresponding to the sgRNA, namely Oligo-F and Oligo-R sequences, annealing the oligonucleotide single-stranded DNA, and connecting the oligonucleotide single-stranded DNA to a BsaI enzyme cutting site of a plasmid pAAV2_ SlutCas9_ U6_ BsaI to obtain pAAV2_ SlutCas9-hU 6-sgRNA;

(3) Delivering a vector expressing the SlutCas9 protein, sgRNA, into a cell containing a target site;

(4) And carrying out PCR amplification on the edited target site, carrying out T7EI enzyme digestion or carrying out second-generation sequencing to detect the editing efficiency.

in the present invention, any targeted sgRNA can be designed for a DNA sequence to be edited according to specific needs, and modifications well known in the art including, but not limited to, phosphorylation, shortening, lengthening, sulfurization, methylation, and hydroxylation can be performed on the sgRNA to some extent.

In the present invention, the CRISPR/slitcas 9 system delivered to the cell may include, but is not limited to, a plasmid expressing a slitcas 9 protein or sgRNA, a retrovirus, an adenovirus, an adeno-associated viral vector or RNA, or a slitcas 9 protein, according to specific needs.

It will be appreciated by those skilled in the art that base N represents A, T, C or G, any of the four bases.

Further, in step (3), the delivery means includes, but is not limited to, liposomes, cationic polymers, nanoparticles, multifunctional envelope-type nanoparticles, and viral vectors.

Further, in step (3), the cells include, but are not limited to, human cells, animal cells, plant cells, bacterial cells, and fungal cells.

Further, in the step (2), the sgRNA has a nucleotide sequence shown in SEQ ID NO. 2, or a nucleotide sequence at least 80% identical to the nucleotide sequence shown in SEQ ID NO. 2, or modifications based on the nucleotide sequence, including but not limited to phosphorylation, shortening, lengthening, sulfurization or methylation.

More specifically, in one embodiment, oligonucleotide single-stranded DNA sequences corresponding to sgrnas, i.e., Oligo-F and Oligo-R, were synthesized as follows:

Oligo-F CACCGCTCGGAGATCATCATTGCG，(SEQ ID NO:4)

Oligo-R AAACCGCAATGATGATCTCCGAGC。(SEQ ID NO:5)。

More specifically, in one embodiment, it can be understood by those skilled in the art that Oligo-F and Oligo-R need to be annealed to become double-stranded DNA, and the annealing reaction system is 1. mu.L of 100. mu.M Oligo-F, 1. mu.L of 100. mu.M Oligo-R, and 28. mu.L of water, and after shaking and mixing, the mixture is placed in a PCR instrument to run an annealing program; the annealing procedure was as follows: 95 ℃ 5min, 85 ℃ 1min, 75 ℃ 1min, 65 ℃ 1min, 55 ℃ 1min, 45 ℃ 1min, 35 ℃ 1min, 25 ℃ 1min, 4 ℃ storage, cooling rate 0.3 ℃/s.

More specifically, in one embodiment, the plasmid pAAV2_ stutcas 9_ ITR requires linearization with BsaI restriction endonuclease (NEB).

More specifically, in one embodiment, the annealed Oligo-F and Oligo-R products are ligated to the linearized pAAV2_ SlutCas9_ ITR backbone vector by DNA ligase.

more specifically, in one embodiment, after transformation of the ligation products into competent cells, the correct clones were verified by Sanger sequencing and the plasmids were extracted for use.

More specifically, in one embodiment, the cell in step (3) is HEK293T comprising a target site having the nucleotide sequence shown in SEQ ID No. 6.

More specifically, in one embodiment, the delivery means in step (3) is a liposome comprising2000 or PEI.

More specifically, in a specific embodiment of the first aspect of the present invention, the template for PCR in the experimental step (4) is edited HEK293T genomic DNA.

More specifically, in one embodiment, the primer sequences amplified by PCR in step (4) are:

F1-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGAGAAAAGCCTTGTTT(SEQ ID NO:7)；

R1-ACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAACTTGTGGCCGTTTAC (SEQ ID NO:8)；

F2-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC(SEQ ID NO:9)；

R2-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTG(SEQ ID NO:10)。

The invention also provides a CRISPR/SlutCas9 system kit for gene editing, which comprises a SlutCas9 protein or sgRNA of a target DNA sequence or a target DNA.

The invention also provides application of the CRISPR/SlutCas9 gene editing system, which comprises gene knockout, site-specific base change, site-specific insertion, gene transcription level regulation, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single-base converter or chromatin imaging tracking.

Drawings

FIG. 1 is a schematic diagram of CRISPR/SlutCas9 gene editing system cutting target DNA. Wherein, the gray oval represents the SlutCas9 protein, the black curved represents the sgRNA sequence, and the darkened region in the upper chain of the genome represents the PAM sequence NNGG.

FIG. 2 is a map schematic diagram of plasmid pAAV2_ SlutCas9_ U6_ BsaI. Among them, the promoter includes AAV2 ITR, CMV enhancer, CMV promoter, SV40 NLS, SlutCas9, nucleoplasmin NLS, 3 XHA, bGH poly (A), human U6 promoter (hU6), BsaI endonuclease site, sgRNA scaffold sequence and other elements.

FIG. 3 shows the result of partial next generation sequencing after the DNA sequence of the target site has been edited. Wherein the editing result has deletion, insertion or mismatch, and the last 4bp represents PAM sequence NNGR.

FIG. 4 shows the cleavage of endogenous site with T7 Endonuclease I. Wherein the arrows indicate the size of the cut fragments.

Detailed Description

The present invention will be further illustrated by the following examples, which are not intended to limit the invention in any way.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. The experimental method not specified for the specific conditions is usually carried out under the conventional conditions or the conditions recommended by the manufacturer.

in a specific embodiment, the CRISPR/stutcas 9 system provided by the invention is a novel gene editing system, method, kit and application thereof.

In a specific embodiment of the invention, the CRISPR/slitcas 9 system is capable of gene editing in a cell, the method comprising the steps of:

1. Construction of plasmid pAAV2_ SlutCas9_ ITR

And (1) downloading an amino acid sequence of the SlutCas9 gene according to the retrieval number A0A1W6BMI2 of the gene on UniProt, wherein the amino acid sequence is shown as SEQ ID NO: 1.

And (2) carrying out codon optimization on the amino acid sequence of the SlutCas9 to obtain a coding sequence highly expressed by the SlutCas9 in human cells, wherein the coding sequence is shown as SEQ ID NO. 11.

And (3) carrying out gene synthesis on the coding sequence of the obtained gene SlutCas9 in a company, and constructing the coding sequence on a pAAV2_ ITR skeleton plasmid to obtain a plasmid pAAV2_ SlutCas9_ ITR, as shown in figure 2.

2. Preparation of linearized plasmid pAAV2_ SlutCas9_ ITR

step (1), carrying out enzyme digestion linearization on the plasmid pAAV2_ SlutCas9_ ITR by using BasI restriction enzyme, wherein an enzyme digestion system comprises: mu.g of plasmid pAAV2_ SlutCas9_ ITR, 5. mu.L of 10 xCetSmart buffer, 1. mu.L of the endonuclease, water to 50. mu.L, and reaction at 37 ℃ for 1 hour.

And (2) carrying out electrophoresis on the product after enzyme digestion on a 1% agarose gel at 120V for 30 minutes.

And (3) cutting off the 7437bp DNA fragment, recovering by using a glue recovery kit according to the steps provided by the manufacturer, and finally eluting by using ultrapure water.

And (4) determining the DNA concentration of the recovered linearized plasmid pAAV2_ SlutCas9_ ITR by using NanoDrop, and reserving or storing at-20 ℃ for long-term storage.

3. Construction of plasmid pAAV2_ SlutCas9-hU6-sgRNA

Designing a gRNA sequence.

Step (2), adding linearized plasmids pAAV2 \uto the sense strand and antisense strand of the designed gRNA sequence pair

The SlutCas9_ ITR is flanked by corresponding cohesive end sequences, and two oligonucleotide single-stranded DNAs are synthesized by the company, wherein the specific sequences are as follows:

Oligo-F CACCGCTCGGAGATCATCATTGCG，(SEQ ID NO:4)

Oligo-R AAACCGCAATGATGATCTCCGAGC。(SEQ ID NO:5)

And (3) annealing the oligonucleotide single-stranded DNA to obtain double-stranded DNA, wherein an annealing reaction system comprises the following steps: after shaking and mixing 1. mu.L of 100. mu. Mooligo-F, 1. mu.L of 100. mu. Mooligo-R and 28. mu.L of water, the mixture was placed in a PCR instrument to run an annealing program: 95 ℃ 5min, 85 ℃ 1min, 75 ℃ 1min, 65 ℃ 1min, 55 ℃ 1min, 45 ℃ 1min, 35 ℃ 1min, 25 ℃ 1min, 4 ℃ storage, cooling rate 0.3 ℃/s.

And (4) connecting the annealed product with the linearized plasmid pAAV2_ SlutCas9_ ITR under the action of DNA ligase according to the steps provided by the product.

Step (5), 1. mu.L of the ligation product was taken for chemical competent transformation, and the growing bacterial clones were subjected to Sanger sequencing validation.

And (6) carrying out sequencing verification on the correctly connected clone shake bacteria, and extracting a plasmid pAAV2_ SlutCas9-hU6-sgRNA for later use.

4. Plasmid pAAV2_ SlutCas9-hU6-sgRNA for transfecting and expressing SlutCas9 protein and sgRNA

Step (1), on day 0, according to transfection needs, the HEK293T cell line containing the sgRNA targeting site is plated in a 6-well plate, the cell density is about 30%, and the sequence of the target site is shown as SEQ ID NO. 6.

Step (2), day 1, transfection was performed in the following transfection system,

i. Adding 2 mu g of plasmid pAAV2_ SlutCas9-hU6-sgRNA to be transfected into 100 mu l of Opti-MEM culture medium, and gently blowing, beating and uniformly mixing;

ii. mixing2000 flick and mix evenly, suck 5 mul and add to 100 mul Opti-MEM culture medium, mix evenly gently, stand 5min at room temperature;

Will dilute2000 and diluted plasmid, gently whipping and mixing, standing at room temperature for 20min, and then adding to the medium of the cells to be transfected.

And (3) placing the cells in a 37 ℃ and 5% CO2 incubator for continuous culture.

5. Preparation of a second Generation sequencing library

Step (1), collecting HEK293T cells after editing for 3 days, and extracting genomic DNA by using a DNA kit according to the steps provided by the product;

step (2), performing first round PCR of PCR library building, performing PCR reaction by using 2xQ5 Mastermix, wherein PCR primers are shown as SEQ ID NO:7-SEQ ID NO:8, and the reaction system is as follows:

The PCR run program was as follows:

And (3) carrying out second round PCR of PCR library building, carrying out PCR reaction by using 2xQ5 Mastermix, wherein the PCR primer is shown as SEQ ID NO: 9-SEQ ID NO:10, and the reaction system is as follows:

The PCR run program was as follows:

and (4) purifying DNA fragments with the size of 366bp by using a gel recovery kit for PCR products of the second round according to the steps provided by the manufacturer, and finishing the preparation of the second generation sequencing library.

6. Analysis of the results of the second generation sequencing

Step (1), the prepared second-generation sequencing library was submitted to the company for paired-end sequencing on HiseqXTen.

step (2) bioinformatics analysis of the next-generation sequencing results, and partial compilation results are shown in FIGS. 2 and 3.

7. Endogenous site validation

Step (1), passing plasmid pAAV2_ SlutCas9-hU6-sgRNA expressing SlutCas9 and sgRNA through2000 were transfected into HEK293T cells according to the manufacturer's protocol, in which,

The sgRNA sequence is: GGCGCAGTTTACTGCACAGGT (SEQ ID NO:12)

the specific sequence of the target site is as follows: GGCGCAGTTTACTGCACAGGTGCGG, respectively; (SEQ ID NO: 13);

Extracting cell genome DNA after 5 days of editing, and amplifying a target DNA sequence by using primers Test-F and Test-R through 2x Q5 Master mix; wherein:

The specific sequence of Test-F is: AGGGAAGAGGAAATGCTGGG (SEQ ID NO:14)

the specific sequence of Test-R is as follows: TGAGCCGCCAGTGTACAGA, respectively; (SEQ ID NO: 15);

step (3), recovering the PCR product through agarose gel, and purifying a DNA fragment with the size of 276 bp;

Step (4), carrying out enzyme digestion on the purified DNA fragment according to the instruction of T7 Endonuclease I, then carrying out gel running detection, wherein the result is shown in figure 4, the left side is a negative control group, and no sgRNA is generated during transfection, and T7 Endonuclease I cuts a targeting sequence and then has no cut fragment, which indicates that no editing is generated; the right panel shows the experimental group, with sgRNA at transfection, and T7 endonucleoclean I cleaved after cleavage of the targeting sequence to show that editing has occurred.

SEQUENCE LISTING

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gtggagaaca acgagggcag acggagcaag agaggagccc ggcggctgaa aagaaggcgg 180

atccaccggc tgaatagaat caagcagctg ctgaaaaacg ccggcctgct ggagggagat 240

gtgctgccta agtctaccaa cccctatgag atcagagtgc ggggcctccg aagccctctg 300

accaaagatg aactggtgat cgccctgctc cacatcgcca aaagaagagg catccacaac 360

atcaacatcg tgggagatga cgaagaaacg gacagcacac tgagtaccac agcccagctg 420

aagaagaacg agaaggctct caagggacag tttgtttgtg aactgcaact ggacagactg 480

gctaatgccc accaagtgcg gggcgagaaa aatcgattta agacagagga cattgtgaag 540

gaagtcagag ccctgcttca gcaacagcag aacttccaca acatcgataa ttctttcgtg 600

gaacagtaca ttgccctgct ggagagccgg aggacctact acgagggccc tggcgaaggc 660

tctccttacg gctgggacgg cgacattaag aagtggtacg agatgctgat gggctattgc 720

acctacttcc ctgaagagct gagaagcgtg aagtacgcct acaccgccga tctgttcaac 780

gccctgaatg acctgaacaa cctggtgatc acccgggacg acaacagcaa attgacatac 840

gccgagaagt accatatcat cgagaacgtg ttcaaacaga agaaagtacc tacactgaag 900

cagatcgcca aggaaatcgg agtgaacgag agcgatatta agggctacag aatcaacaaa 960

tctgagaaac ctctgttcac cagcttcaaa ctgtaccacg atctgaagag cgtgttcagc 1020

gaccctacaa aactggaaga tatcgacttg ctggaccgaa tcgccgtggt gctgaccatg 1080

taccaagatg ccgaatccat gaaagccgcc ctgaacacct tccctgaagt gttcagcgaa 1140

gcagagaaag agaagctgag cgccctcaca ggctacgctg gcacccatag actgtctctg 1200

aagtgcatga acctgctgat ccctgatctg tggcagacaa gcctgaacca gatggaactg 1260

ttcgtgaagc tgaatctgaa accacagaag ctggacctga gccagtgcca ccagattcct 1320

acccagctgg tggacgagtt catcctgtct cctgtggtga aaagagcctt cacccaaagc 1380

atcaaggtga tcaacgccat catccagaaa tacggcctgc ccgacgacat cataatcgag 1440

ctggccaggg aaaaaaacag cgccgataag cggaagttcc tgaatcagct gcagaagaag 1500

aacgagaagg cccggcacga gatcaatacc ctggtggccc agtacggcca gccaaatgct 1560

aagcggctgg tggaaaagat cacactgcac cagcagcagg agggcaagtg tctgtactcc 1620

ctgaaggata tccccctgga gcagctgctg aagcagccct acctgtacga ggtggaccac 1680

atcatcccca gaagcgtttc tttcgacaac agcatgcaga acaaggtgct ggtcctggcc 1740

gaagaaaacg ccaaaaaggg caaccagacc ccttaccagt acctgaatag cagagaggcc 1800

agcatgacct accccgaatt caaacagcac atcctgaatc tgagcaaggc caaggaccgg 1860

atcagcaaga agaagcggaa ctacctgctc gaggaaagag atatcaacaa gttcgacgtg 1920

caaaaggact tcatcaacag aaacctggtt gacaccagat acgccaccag agagctcgcc 1980

tctctgctga aggcttattt caagacacat gagctccctg tgaaagtgaa gaccatcaac 2040

ggcggattta cccactacct gagaaaggtg tggaagtttg acaaagatag aaacaagggc 2100

tacaagcacc acgccgagga tgcactgatc atcgccaacg ccgactttct gtttaagaac 2160

cagactctga acaaaatcga ggctatcctg aacgagcccg gcagagaggt ggaatctgac 2220

acagtgaagg tgcagagcga ggacaattac caggacctgt ttgagaacac caagaaggct 2280

ttcgccatca agaatttcaa ggatttcaag tttagccaca gagtggacca gaagcctaac 2340

cggcagctcg tgaacgacac cctgtacagc accagagagg tgaacgaaga tctgtacgtg 2400

gtgcagaccc tgaaggacat ctacagcaag gacaacaaag acgtgaagcg gctcttcgac 2460

aagcaacccg agaagttcct gatgttccag cacgaccccg agacattcaa gaaattcgag 2520

ctggctatga agcaatatgc cgaagagaag aaccctctgg ctagatacta cgaggaacag 2580

ggctacatca ccaagtacgc caagaaagga gacggcccac ctgtgaaaag cctcaaatac 2640

atcggcaaga aggtgggaaa acacctggat gtgaccggcg actacgagga tagcaaccgg 2700

aagctggtga agctgagcct gaagtccttt agattcgata tctaccacac agacaagggc 2760

tacaagatgg ttccaatcac ttacctggat gtgcagaaaa aagagaagta ctactacatc 2820

cccaccgaga aatacgaagc cctgaagcag gagaagggca tcaaccagaa tgctcagttc 2880

attggcagct tctactacaa cgacctgatc gagttcgatg gcgagctgta tagagtgatc 2940

ggcatcaaca acggcgacaa gaatctcgtt gaactcgaca tggtggacat tagatataag 3000

gaatactgcg agctgaactc catcaccacc acacctagaa tcgttaagac catcagcccc 3060

aagacccaga gcatcgagaa gtacacaaca gatatcctgg gcaacctgta caaagcccag 3120

cctggcaaga agcctcagtt catcttcaac aaagacgagg at 3162

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<400> 12

ggcgcagttt actgcacagg t 21

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence

<400> 13

ggcgcagttt actgcacagg tgcgg 25

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

agggaagagg aaatgctggg 20

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence

<400> 15

tgagccgcca gtgtacaga 19

Claims (18)

1. CRISPR/Cas9 gene editing linethe CRISPR/Cas9 system is used for gene editing in cells or in vitroSlutCas9The protein and sgRNA complex can accurately position a target DNA sequence and generate cutting, so that double-strand break damage occurs to DNA; the above-mentionedSlutCas9The protein has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence which is at least 80 percent identical to the amino acid sequence shown in SEQ ID NO. 1; the sgRNA has a nucleotide sequence shown in SEQ ID NO. 2, or a sgRNA sequence modified based on SEQ ID NO. 2.

2. the CRISPR/Cas9 gene editing system of claim 1, wherein the cells comprise eukaryotic cells and prokaryotic cells; the eukaryotic cells include mammalian cells and plant cells; the mammalian cell includes a Chinese hamster ovary cell, a baby hamster kidney cell, a mouse Sertoli cell, a mouse mammary tumor cell, a buffalo rat liver cell, a rat liver tumor cell, a monkey kidney CVI line transformed by SV40, a monkey kidney cell, a canine kidney cell, a human cervical cancer cell, a human lung cell, a human liver cell, an HIH/3T3 cell, a human U2-OS osteosarcoma cell, a human A549 cell, a human K562 cell, a human HEK293T cell, a human HCT116 cell, or a human MCF-7 cell or a TRI cell.

3. The CRISPR/Cas9 gene editing system according to claim 1, wherein the CRISPR/Cas9 gene editing systemSlutCas9Proteins comprising no cleavage activity or having only single strand cleavage activity or having double strand cleavage activitySlutCas9A protein.

4. the CRISPR/Cas9 gene editing system according to claim 1, wherein the precisely positioned DNA sequence comprises a sequence of 20bp or 21bp at the 5' end of sgRNA which can form a base complementary pairing structure with a target DNA sequence.

5. The CRISPR/Cas9 gene editing system according to claim 1, wherein the precisely located targeting DNA sequence comprisesSlutCas9protein and sgRNA complex recognitiontarget PAM sequences on the DNA sequence.

6. The CRISPR/Cas9 gene editing system according to claim 5, wherein the PAM sequence is NNGR and the targeting DNA sequence is SEQ ID NO 3.

7. The CRISPR/Cas9 gene editing system according to claim 1, wherein the sgRNA can be phosphorylated, shortened, lengthened, sulfurized, methylated, or hydroxylated modified.

8. The CRISPR/Cas9 gene editing system according to claim 1, wherein the CRISPR/Cas9 gene editing systemSlutCas9Protein and sgRNA complex can precisely target DNA sequenceSlutCas9The complex of protein and sgRNA can recognize and bind to a specific DNA sequence, or to be bound toSlutCas9Other proteins of the protein fusion or proteins that specifically recognize the sgRNA are brought into position to target the DNA.

9. The CRISPR/Cas9 gene editing system according to claim 8, wherein the CRISPR/Cas9 gene editing systemSlutCas9Protein and sgRNA complexes or withSlutCas9Other proteins of the protein fusion or proteins that specifically recognize the sgrnas can modify and regulate targeted DNA regions, including regulation of gene transcription levels, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single base converters, or chromatin imaging tracking.

10. the CRISPR/Cas9 gene editing system according to claim 9, wherein the single base switch comprises a switch between bases adenine to guanine, cytosine to thymine, cytosine to uracil or other bases.

11. CRISPR/according to one of claims 1 to 10SlutCas9A method for gene editing in a cell by a gene editing system comprisingSlutCas9Compound recognition and localization targeting DN of protein and sgRNAA, editing DNA; finally, detecting the editing efficiency; the method comprises the following specific steps:

(1) Synthetic humanizationSlutCas9A gene sequence; and cloning the gene into an expression vector to obtain pAAV2 \ uSlutCas9_ITR；

(2) Single-stranded oligonucleotides DNA corresponding to sgRNA, i.e., Oligo-F and Oligo-R sequences, were synthesized, annealed and ligated to plasmid pAAV2 \\ uSlutCas9BsaI cleavage site of U6 BsaI to obtain pAAV 2USlutCas9-hU6-sgRNA；

(3) Will expressSlutCas9The protein, vector of sgRNA, is delivered into a cell containing the target site;

(4) And carrying out PCR amplification on the edited target site, carrying out T7EI enzyme digestion or carrying out second-generation sequencing to detect the editing efficiency.

12. The method of claim 11 wherein said pAAV2 \uSlutCas9-hU6-sgRNA is an adeno-associated virus backbone plasmid comprising AAV2 ITR, CMV enhancer, CMV promoter, SV40 NLS, SlutCas9, nucleoplasmin NLS, 3 xha, bGH poly (a), human U6 promoter, BsaI endonuclease site, sgRNA scaffold sequence.

13. The method of claim 11, wherein the CRISPR ∑ is delivered to a cellSlutCas9The system includes an expressionSlutCas9Plasmids, retroviruses, adenoviruses, adeno-associated viral vectors or RNAs or of proteins or sgRNAsSlutCas9A protein.

14. the method of claim 11, wherein the sequences of single-stranded oligonucleotides corresponding to sgrnas, i.e., Oligo-F and Oligo-R, are shown in SEQ ID NOs 4 and 5, are synthesized.

15. The method according to claim 11, wherein the target site of the cell in step (3) has the nucleotide sequence shown as SEQ ID NO 6.

16. The method according to claim 11, wherein the template for PCR in step (4) is edited DNA; the primer sequences for PCR amplification are: SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO 10.

17. CRISPR/according to one of claims 1 to 10SlutCas9A kit for a gene editing system, the kit comprisingSlutCas9sgRNA or targeting DNA of a protein or targeting DNA sequence.

18. CRISPR/according to one of claims 1 to 10SlutCas9applications of gene editing systems, including gene knockout, site-directed base alteration, site-directed insertion, regulation of gene transcription levels, regulation of DNA methylation, DNA acetylation modification, histone acetylation modification, single base converters, or chromatin imaging tracking.