CN111471745B - DNA targeting capture method based on CRISPR/Cas9 system mediation - Google Patents

Abstract

The invention discloses a CRISPR/Cas9 system-mediated DNA targeted capturing method, which comprises the steps of designing a sgRNA respectively for the upstream and downstream of a target DNA region, adding different capturing sequences and a sequence which is reversely complementary with the sgRNA at the 3' ends of two sgRNA sequences to form a pegRNA, inserting different capturing sequences respectively for the upstream and downstream of the target DNA region through pegRNA-mediated fixed-point insertion when a pegRNA-Cas9 protein compound is combined with the target DNA in a targeted manner, carrying out PCR amplification through a primer containing the capturing sequences, carrying out targeted enrichment and separating the target DNA from a DNA library or a whole genome, and directly using the target DNA for subsequent capture sequencing. Compared with other enrichment methods, the method provided by the invention is simple, rapid, strong in specificity, high in sensitivity and has wide application advantages in DNA sequence analysis.

Description

DNA targeting capture method based on CRISPR/Cas9 system mediation

Technical Field

The invention belongs to the technical field of gene sequencing, and particularly relates to a DNA targeting capture method based on CRISPR/Cas9 system mediation.

Background

In recent years, there has been much interest in DNA sequence information for specific regions, and DNA sequence analysis can discover genetic and epigenetic changes in the genome, such as Single Nucleotide Variations (SNV), Copy Number Variations (CNV), translocations and methylation. With the advancement of DNA sequencing technology, Next Generation Sequencing (NGS) provides a powerful tool to decode DNA information at genome scale and single base resolution. NGS-based DNA analysis has a variety of applications, such as precision medicine implemented on humans and SNP breeding employed in animals.

CRISPR-Cas9 is an adaptive immune defense formed during long-term evolution of bacteria and archaea, and can be used to fight invading viruses and foreign DNA. The CRISPR-Cas9 gene editing technology is a technology for carrying out specific DNA modification on a target gene, and is also a method used for the leading edge of gene editing at present. David Liu et al improved the existing gene editing CRSPR/cas9 technology, proposed the concept of prime editing, and the novel gene editing technology "prime editing" was able to directly edit human cells in an accurate, efficient and highly versatile manner. This approach expands the range of gene editing for biological and therapeutic studies and makes it possible to correct up to 89% of known disease-causing genetic variations. The technology can realize the accurate insertion of a target sequence and can effectively reduce off-target effects, and the proposal of the technology enables the capture of the target sequence to be simple and efficient.

At present, target sequencing methods related to a target region have many methods, but all the methods have certain limitations, for example, a hybridization capture method is used, firstly, a genome is broken by ultrasound, hybridization is carried out in a DNA-RNA mode and the like, then, a probe captures a target DNA, PCR enrichment is carried out, and the hybridization-based sequencing method has the main defects that a genome fragment depending on ultrasonic treatment can generate a DNA fragment with random size, certain preference is generated in the PCR enrichment process, a short fragment can be preferentially amplified, so that sequencing reads are not uniform, partial sequence information in a long fragment is lost and the like; in addition, there is a CRISPR-Cas9 system-based capture method, such as the CRISPR-DS technology developed by nachmann D, which can realize high-sensitivity detection, but its limitations are: after the capture is finished, the A tail and the ligation reaction are required to be added, then PCR enrichment is carried out, the experimental flow is complex, and in addition, the success rate of the A tail and the ligation step may influence the capture success rate.

The invention utilizes the novel gene editing technology 'prime editing' to effectively edit and insert a target region at a fixed point, thereby efficiently and accurately capturing the target region, effectively enriching the target region through PCR amplification, finally carrying out high-throughput sequencing and analysis on the target region, effectively reducing the sequencing cost, improving the specificity and sensitivity of capture, and having strong practicability in the fields of DNA sequence analysis and the like. Compared with the prior art, the method directly performs PCR enrichment after Cas9 is cut, reduces the error rate by simplifying the experimental steps, and can realize the capture of the DNA sequence at the fastest speed.

Disclosure of Invention

The invention aims to provide a CRISPR/Cas9 system-mediated DNA targeted capture method, which is used for simply and quickly realizing DNA sequence analysis by means of an NGS (Next Generation System) sequencing method through directly inserting a sequencing joint into a target region.

In order to achieve the purpose, the invention adopts the following technical scheme:

the DNA targeting capture method based on CRISPR/Cas9 system mediation specifically comprises the following steps: designing a sgRNA aiming at the upstream and downstream of a target DNA region, adding different capture sequences and a sequence reversely complementary with the sgRNA at the 3' ends of two sgRNA sequences to form a pegRNA, inserting different capture sequences into presgRNA combined with Cas9 protein at the upstream and downstream fixed points of the target DNA, and amplifying the target DNA by a primer containing the capture sequences, thereby carrying out targeted capture and separation on the target DNA.

Wherein, the capture sequence can be any DNA sequence, preferably a part of i5 and i7 sequencing joints in NGS sequencing, for example, the universal sequence inserted in the upstream region of the target DNA is named as RT1, and the sequence is: 5'-GGAATCGCACCAGCGTGT-3', the general sequence inserted in the downstream region of the target DNA was named RT2 with sequence 5'-CACTGCGGCTCCTCATCC-3'.

Among them, Cas9 proteins include the conventional Cas9 protein as well as various Cas9 proteins formed through other engineering processes.

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

1. the genome is directly enriched by PCR without additional interruption and hybridization capture, so that the capture accuracy is improved, false positive in the capture process is reduced, and the capture efficiency is improved;

2. the method has the advantages of simple technical operation and few reaction steps, and can be popularized and used on a large scale.

Drawings

FIG. 1: CRISPR/Cas9 system-mediated DNA targeted capture principle

As shown in fig. 1, two different pegrnas are designed, after in vitro transcription, a pegRNA-Cas9 complex is formed with Cas9 protein, and then a corresponding sequencing linker is reverse transcribed in a target region directly through co-incubation with a substrate gDNA and pegRNA-mediated site-directed insertion, and a capture product is recovered through PCR enrichment.

FIG. 2: electrophorograms before and after Cas9 enzyme digestion of Myod vector

As shown in the figure, M1:100bp DNA Ladder is composed of 11 linear double-stranded DNA bands, the fragments are 1500bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp and 100bp sequentially, M2: DL2000 DNA Marker consists of 6 double-stranded DNAs of 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp. And (3) after Cas9 enzyme digestion, recovering: the DNA refers to a fragment recovered after a Cas9 enzyme cuts a Myod vector and is precipitated by isopropanol, and as can be seen from the figure, the expected capture fragment 701bp is generated after the vector is cut by two pegRNA, and NC refers to an original fragment formed by connecting an original Myod gene and a T vector.

FIG. 3: after digestion, gel was recovered and electrophorograms were amplified using RT1-F and RT2-R

As shown, the PCR amplification products are represented as: the product precipitated by isopropanol is used as a template, RT1-F, RT2-R is used as an amplification product of a primer, and the expected capture fragment 701bp is generated after the product is enriched by PCR.

FIG. 4: alignment chart of sequencing results

As shown in the figure, the results of alignment of different single clones with the original Myod fragment after ligation of the PCR product with the T vector, from the sequencing results, yielded the expected two RT1, RT2 inserts.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples, and the techniques used in the following examples are conventional techniques known to those skilled in the art unless otherwise specified; the instruments, reagents and the like used are all available to those skilled in the art from public sources such as commercial sources and the like.

Example (b): targeted capture of mouse Myod gene target region using CRISPR/Cas9 mediation

1.1 preparation of pegRNA for targeting mouse Myod Gene target DNA region

1.1.1 through NCBI database, determining and downloading mouse gene Myod gene sequence, using Chop-Chop online sgRNA design software (http:// chopchopchop. cbu. uib. no /), according to the specificity evaluation result (see Table 1 below), selecting the best sgRNA (containing PAM sequence) on the sense strand and the antisense strand of the target region respectively.

The sgRNA sequence on the sense strand is sgRNA 2: 5'-GCACCACCGTAGGCACTCCTGGG-3'

The sgRNA sequence on the negative strand is sgRNA 4: 5'-GTCGTAGCCATTCTGCCGCCGGG-3'

Table 1 design and evaluation of sgrnas targeting mouse Myod gene using software

1.1.2 preparation of PegRNA

Designing an in vitro transcription template according to the generated sgRNA

First, two pegRNA primers for annealing were designed separately

presgRNA2-F:

5′TAATACGACTCACTATAGGGCACCACCGTAGGCACTCCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA-3′

presgRNA2-R:

5′gcatgcaggcctctgcagAAAAAAAAACCGTAGGCACTCCACTGCGGCTCCTCATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGG-3′

presgRNA4-F：

5′TAATACGACTCACTATAGGGTCGTAGCCATTCTGCCGCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA-3′

presgRNA4-R：

5′gcatgcaggcctctgcagAAAAAAAAAGCCATTCTGCCGACACGCTGGTGCGATTCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGG-3′

Design of PCR amplification primers

p-sg-F:5′-GGATCCTAATACGACTCACTA-3′

KZ-2-Cas9-pegRNA-R:5′-gcatgcaggcctctgcagAAA-3′

And (3) PCR reaction system:

and (3) PCR reaction conditions:

the PCR product was electrophoresed in a sample, purified by a gel recovery kit, and stored at-20 ℃ for in vitro transcription.

1.1.3 in vitro transcription System:

reaction conditions are as follows: incubation at 37 ℃ overnight

1.1.4 genome removal

Reaction conditions are as follows: 30min at 37 DEG C

1.2 Assembly CRISPR/Cas9-presgRNA Complex

Reaction conditions are as follows: 15min at 25 DEG C

1.3 digestion of the target region System

Reaction conditions are as follows: 120min at 37 DEG C

85℃15min

1.4 recovery of the cleavage product

After the enzyme digestion is finished, adding 4 mu l of RNase into the system, incubating for 30min at 37 ℃, adding proteinase K, treating for 6-8h at 37 ℃ and 800rpm, and degrading residual RNA in the system and unreacted Cas9 enzyme completely. The digested complex was resinated and subjected to isopropanol precipitation overnight. The recovered product was frozen at-20 ℃.

1.5 PCR enrichment of predicted fragments

1.5.1 PCR System

1.5.2 PCR procedure

1.6 TA clone validation

And (3) carrying out electrophoresis on the PCR, carrying out gel recovery on a target fragment, and carrying out TA connection on a product recovered from the gel in a TA connection reaction system:

reaction conditions are as follows: 5min at 37 DEG C

After the reaction is finished, the ligation product is transformed into an escherichia coli cell (Novozam, C505-02), after overnight culture at 37 ℃, a proper amount of monoclonal antibody is picked for sequencing, and the sequencing result is compared and verified with a reference genome. The sequence of Myod connected with T carrier is shown as SEQ ID NO.1, the sequence of PCR product connected with T carrier is shown as SEQ ID NO.2, the sequencing comparison result shows that two different sequencing joints are successfully and directionally inserted into the target DNA region of Myod gene, the target sequence to be targeted is successfully separated, the sequence of PCR targeted can be directly sequenced on NGS machine without additional treatment to the sample, and the result proves that the technology is simple, rapid, strong in specificity and high in sensitivity. Therefore, we provide a new powerful tool for targeted sequencing, with obvious advantages compared with other enrichment methods at present.

Sequence listing

<110> university of agriculture in Huazhong

<120> DNA targeting capture method based on CRISPR/Cas9 system mediation

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gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 60

tgataccgct cgccgcagcc gaacgaccga gcgcagcgag taagtgagcg aggaagcgga 120

agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 180

gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 240

gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 300

aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 360

cagaattaac cctcactaaa ggtactagtc ctgcaggttt aaacgaattc gcccttgagt 420

gcctacggtg gtgcgccctc tgctgctgca gtcgatctct caaagcacct gataaatcgc 480

attggggttt gagcctgcag gacactgagg ggcggcgtcg ggagacgggg tctgggttcc 540

ctgttctgtg tcgcttaggg atgccccctc tggcggaccc ggaggcgact ctggtggtgc 600

atctgccaaa agcagcgcag gcgcagcggg gctgtctgtg gagatgcgct ccactatgct 660

ggacaggcag tcgaggctcg acacagccgc actcttccct ggcctggact ctgtgagggg 720

agtggaaggt tagtaatcca aacttgcggc ccttctcaag tctcctgccc ataggcacgg 780

agctttgtag caaaagggaa actgaggact tgctagactg gaaggagaaa ggagaggaag 840

gtgtgcatca tagttctttg tcccctcccc caatgctccc ccccagcctc ccccccaccc 900

cccaccatcc caaggtacgg gtatgtatgt ggtggaggca ggaagaggat actaagccta 960

gggagcgagg aaagtttcaa gggggacaca gaaggccgat aaaggcctgc tagctgggaa 1020

cagctgagaa tacgcaccgc gcaccgcctc actgtagtag gcggtgtcgt agccattctg 1080

ccgaagggcg aattcgcggc cgctaaattc aattcgccct atagtgaatc gtattacaat 1140

tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 1200

cgccttgcag cacatccacc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 1260

cgcccgtccc aacagttgcg cagcctatac gtacggcagt ttaaggttta cacctataaa 1320

agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgccgggg 1380

cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa 1440

ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc 1500

agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac 1560

atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggcat gagattatca 1620

aaaagcatct tcacctagat cgttttcacg tagaaagcca gtccgcagaa acggtgctga 1680

ccccggatga atgtcagcta ctgggctatc tggacaagag aaaacgcaag cgcaaagaga 1740

aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca 1800

gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa 1860

gtaaactgga tggctttctc gccgccaagg atctgatggc gcaggggatc aagctctgat 1920

caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct 1980

ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 2040

tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 2100

gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc 2160

acgacgggcg ttccatgcgc agctgtgctc gacgttgtca ctgaagcggg aagagactgg 2220

ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 2280

aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 2340

ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 2400

cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 2460

gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 2520

tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 2580

ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 2640

cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 2700

cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaattattaa cgcttacaat 2760

ttcctgatgc ggtattttct cgttacgcat ctgtgcggta tttcacaccg catacaggtg 2820

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 2880

atatgtatcc gctcatgaga ttatcaaata ggatcttcac ctagatcctg ttaaattaaa 2940

aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 3000

gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 3060

gactacccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 3120

caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 3180

ccggaagagc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 3240

attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 3300

ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 3360

gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 3420

ctttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 3480

tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 3540

gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 3600

cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 3660

gaaaacgttc ttcggggcga aaactctcaa gaatcttacc gctgttgaga tccagttcga 3720

tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 3780

ggtgagcaaa aacaggaagg caaaatgccg caaaaaacgg aataagagcg acacggaaat 3840

gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 3900

tcatgaccaa aatccgttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 3960

agatcaaacg atcttcttga gatcgttttt ttctgcgcgt aatctgctgc ttgcaaacaa 4020

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 4080

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtcgttcta gtgtagccgt 4140

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4200

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gtctcaagac 4260

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4320

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4380

ccacgcttcc cgaagcgaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4440

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4500

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4560

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggg cttttgctgg ccttttgctc 4620

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gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 60

tgataccgct cgccgcagcc gaacgaccga gcgcagcgag taagtgagcg aggaagcgga 120

agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 180

gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 240

gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 300

aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 360

cagaattaac cctcactaaa ggtactagtc ctgcaggttt aaacgaattc gcccttagcg 420

tctttatttc caacacctga ctcgcccgct tgaggaataa acatacaaag tttttgttgc 480

actacacagc atgcctggga gataaatata gcccaagggg ctcccgctct ggccaagcaa 540

ctcttattta cacgaataga ttacaaaagt cttatataaa aaaataagtg gtcctgaacc 600

gcgggaaggg ggagagtggg gtatggttac acctgttaca cctgagacct gagtggcctc 660

cgcaagctgt ggggaaaagt gggtgccagg agggctccag aaagtgacaa acaaaaggat 720

gtggagggca gggcttaggt gtgcaaggct cacccctgtg ctatgaggaa aggaagagcg 780

cctggccact caaggatcag ctctggccca gtgaggtgca gccagagtgc aagtggcctt 840

cgctgtgagc cgcatgaaaa aacaaacaaa caactgaagg actacaacaa caacaacaaa 900

aaggactatg tcctttcttt ggggctggat ctaggacaga cttctgctct tcccttccct 960

ctggaagaac ggcttcgaaa ggacagttgg gaagagtgtc atttaagctt catcttttgg 1020

gcgtgaagaa ccaggggcac catccccagg agtgcctacg gtggtgcgcc ctctgctgct 1080

gcagtcgatc tctcaaagca cctgataaat cgcattgggg tttgagcctg caggacactg 1140

aggggcggcg tcgggagacg gggtctgggt tccctgttct gtgtcgctta gggatgcccc 1200

ctctggcgga cccggaggcg actctggtgg tgcatctgcc aaaagcagcg caggcgcagc 1260

ggggctgtct gtggagatgc gctccactat gctggacagg cagtcgaggc tcgacacagc 1320

cgcactcttc cctggcctgg actctgtgag gggagtggaa ggttagtaat ccaaacttgc 1380

ggcccttctc aagtctcctg cccataggca cggagctttg tagcaaaagg gaaactgagg 1440

acttgctaga ctggaaggag aaaggagagg aaggtgtgca tcatagttct ttgtcccctc 1500

ccccaatgct cccccccagc ctccccccca ccccccacca tcccaaggta cgggtatgta 1560

tgtggtggag gcaggaagag gatactaagc ctagggagcg aggaaagttt caagggggac 1620

acagaaggcc gataaaggcc tgctagctgg gaacagctga gaatacgcac cgcgcaccgc 1680

ctcactgtag taggcggtgt cgtagccatt ctgccgccgg gggccgcttg gggggccgct 1740

gtaatccatc tgcgaatagc aaggataaca gagttaggtc tacagggccc gagtgggtct 1800

caagcaacac tccttgtcat cacccctccc tcactccctt agtctcagct gctggttcca 1860

ggactgggtc agccccaggt gctaggagcc agccacacac tggaagctgt cgcatgcggg 1920

gccaaactga gagtggacag tcactcagga acaataatct gcattagggt aataatgaga 1980

aggaaaagca tatcccgggg tctgcgcagc agtgttccac ccccaatcgg cagaatcata 2040

gatgccctgg gtctccaaag cgactccagt gtcagaaagg gcgaattcgc ggccgctaaa 2100

ttcaattcgc cctatagtga atcgtattac aattcactgg ccgtcgtttt acaacgtcgt 2160

gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc acctttcgcc 2220

agctggcgta atagcgaaga ggcccgcacc gatcgcccgt cccaacagtt gcgcagccta 2280

tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg 2340

gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt 2400

gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat 2460

gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa 2520

gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc 2580

tggggaatat aaatgtcagg catgagatta tcaaaaagca tcttcaccta gatcgttttc 2640

acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct 2700

atctggacaa gagaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca 2760

tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg 2820

gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt ctcgccgcca 2880

aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc 2940

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 3000

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 3060

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 3120

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccatg cgcagctgtg 3180

ctcgacgttg tcactgaagc gggaagagac tggctgctat tgggcgaagt gccggggcag 3240

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 3300

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 3360

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 3420

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 3480

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 3540

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 3600

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 3660

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 3720

gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt tctcgttacg 3780

catctgtgcg gtatttcaca ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa 3840

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agattatcaa 3900

ataggatctt cacctagatc ctgttaaatt aaaaatgaag ttttaaatca atctaaagta 3960

tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4020

cgatctgtct atttcgttca tccatagttg cctgactacc cgtcgtgtag ataactacga 4080

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 4140

cggctccaga tttatcagca ataaaccagc cagccggaag agccgagcgc agaagtggtc 4200

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4260

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4320

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4380

gatcccccat gttgtgcaaa aaagcggtta gctctttcgg tcctccgatc gttgtcagaa 4440

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4500

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4560

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4620

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4680

caagaatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4740

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4800

ccgcaaaaaa cggaataaga gcgacacgga aatgttgaat actcatactc ttcctttttc 4860

aatattattg aagcatttat cagggttatt gtctcatgac caaaatccgt taacgtgagt 4920

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa acgatcttct tgagatcgtt 4980

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5040

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5100

agataccaaa tactgtcgtt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 5160

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 5220

ataagtcgtg tcttaccggg ttggtctcaa gacgatagtt accggataag gcgcagcggt 5280

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5340

tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaagcg agaaaggcgg 5400

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5460

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5520

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 5580

tacggttcct gggcttttgc tggccttttg ctcacat 5617

Claims (4)

1. A CRISPR/Cas9 system-mediated DNA targeted capturing method is characterized in that a sgRNA is designed for the upstream and downstream of a target DNA region, different capturing sequences and a sequence which is reversely complementary with the sgRNAs are added to the 3' ends of two sgRNA sequences to form two corresponding pegRNAs, the pegRNA combined with Cas9 protein inserts different capturing sequences into the upstream and downstream fixed points of the target DNA, and the target DNA is amplified through a primer containing the capturing sequences, so that the target DNA is captured and separated in a targeted mode.

2. The CRISPR/Cas9 system-based mediated DNA targeted capture method of claim 1, wherein the Cas9 protein comprises a conventional Cas9 protein and various Cas9 proteins formed by other engineering processes.

3. The CRISPR/Cas9 system-based mediated DNA targeted capturing method as claimed in claim 1 or 2, wherein the general sequence inserted in the upstream region of the target DNA is named as RT1 with sequence 5'-GGAATCGCACCAGCGTGT-3', and the general sequence inserted in the downstream region of the target DNA is named as RT2 with sequence 5'-CACTGCGGCTCCTCATCC-3'.

4. The CRISPR/Cas9 system-based mediated DNA targeted capture method according to claim 3, wherein the amplification primer of the target DNA is RT 1-F: 5'-GGAATCGCACCAGCGTGT-3', RT 2-R: 5'-GGATGAGGAGCCGCAGTG-3' are provided.

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