CN111876422B - Screening report system capable of being used for enriching CRISPR/Cas9 mediated accurate NHEJ repair cells - Google Patents

Abstract

The invention discloses a screening report system for enriching CRISPR/Cas9 mediated accurate NHEJ repair cells, which comprises an accurate NHEJ repair screening report vector and a long fragment targeting vector of a cell genome co-transfected with the screening report vector in a target editing cell, wherein the accurate NHEJ repair screening report vector comprises a screening report gene expression cassette and two sgRNA expression cassettes, a transcription region of the screening report gene expression cassette comprises a double sgRNA target site construction sequence for being inserted into a selected partition position of a drug screening gene sequence, and the double sgRNA target sites respectively take sgRNAs transcribed by the two sgRNA expression cassettes as guide sequences of the targeting screening report gene expression cassette. The screening report system of the invention can efficiently enrich positive cell clones with accurate deletion of target gene long fragments by cotransfection and drug screening.

Description

Screening report system capable of being used for enriching CRISPR/Cas9 mediated accurate NHEJ repair cells

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a construction method of a screening report vector for enriching CRISPR/Cas9 mediated accurate NHEJ repair cells and application of the screening report vector in accurate deletion of long fragments of cell genome.

Background

Accurate editing can safely modify genome, and is becoming an important means for researching gene functions and cultivating new animal varieties. The CRISPR/Cas9 technology can modify a target gene more simply, conveniently and efficiently, and in the CRISPR/Cas9 system, the Cas9 nuclease can perform targeted recognition on a target genome locus under the guidance of single-stranded guide RNA (sgRNA) to induce double-strand breaks (DSBs) of DNA. Double strand break Repair (Repair) of DNA has two major pathways: non-homologous end joining (NHEJ) and homologous recombination (HDR). Classical NHEJ is considered an error-prone DSBs repair pathway, generally resulting in random insertions or deletions (indels) of the target site. And HDR is more suitable for being applied to accurate editing such as single base substitution, target fragment knock-in and knock-out and the like.

Classical NHEJ pathway mediated deletion of gene fragments has been the preferred way to delete genes, but due to their small index generation, the deleted genotypes are not accurately predicted and set. Thus, classical NHEJ-mediated gene deletion individuals present a degree of genetic safety issues. Although the HDR-mediated deletion of the donor-dependent gene fragment can achieve the purpose of accurate editing, the efficiency of HDR repair is far lower than that of NHEJ repair in practical application, and the repair efficiency of HDR is reduced along with the extension of the length of editing fragments (donor integration), so that the HDR repair is difficult to be widely applied.

Recent studies have found a DNA double-strand break repair approach different from the classical NHEJ pathway, which directly connects the break nicks by DNA ligase without digestion and modification at the free ends of the DNA when Cas9 nuclease causes double-strand break of the DNA. This repair is called exact NHEJ repair (exact-NHEJ, abbreviated acNHEJ). Repair of acNHEJ is accurate and predictable, and repair efficiency of acNHEJ is almost the same as classical NHEJ. However, the length of the acNHEJ repair mediated precise fragment deletion is only within 100bp, and no report is made on the acNHEJ mediated precise deletion of long fragments.

CRISPR/Cas9 in combination with NHEJ-mediated repair of DSBs is commonly used in gene deletion studies. However, with the continued innovation of gene editing technology, research applications for gene editing on animal individuals have begun to shift to safe, precise directions. Therefore, whether or not the target gene can be edited efficiently and accurately becomes a standard as to whether or not the gene editing tool can be widely used. At present, the technical problems to be solved are as follows: the efficiency of accurate deletion of the long gene fragment is improved, and a corresponding gene editing/screening report system is developed.

Disclosure of Invention

The invention aims to provide a screening report system for enriching CRISPR/Cas9 mediated accurate NHEJ repair cells.

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

an accurate NHEJ repair recombination screen gene comprising a double sgRNA target site construct sequence for insertion at a selected cut-off position of a screening/reporter gene (e.g., drug screen gene) sequence (the cut-off position is located between two bases within the drug screen gene sequence, the drug screen gene is recombined by insertion of the double sgRNA target site construct sequence and expression of the drug screen gene is blocked), the double sgRNA target site construct sequence comprising a three base sequence for constituting two sgRNA sequences with a gene (e.g., drug screen gene) target sequence located upstream and downstream of the cut-off position and two PAM sequences linked to the corresponding sgRNA sequences, respectively.

Preferably, the double sgRNA target site building sequence further comprises an unrelated sequence (without any target sequence) for linking the two PAM sequences.

Preferably, the unrelated sequence is a short fragment of 130-150 bp.

The precise NHEJ repair screening report vector comprises a screening report gene expression cassette and two sgRNA expression cassettes, wherein a transcription region of the screening report gene expression cassette comprises a fluorescent report gene sequence and a precise NHEJ repair recombination screening gene sequence constructed by utilizing a drug screening gene (and the double sgRNA target site construction sequence), and the double sgRNA target sites respectively take sgRNAs transcribed by the two sgRNA expression cassettes as guide sequences of the targeting precise NHEJ repair recombination screening gene sequence.

Preferably, the precise NHEJ repair screen reporter vector, e.g., in transfected editing cells of interest, undergoes precise NHEJ repair by cleavage of the CRISPR/Cas9-mediated reporter vector itself (specifically, the precise NHEJ repair recombination screen gene sequence) double sgRNA target site, reverts (i.e., removes the partition by precise deletion of the double sgRNA target site building sequence) in the transcribed region of the screen reporter expression cassette, and allows the precise NHEJ repair screen reporter vector to express corresponding drug resistance.

Preferably, the fluorescent reporter sequence is co-expressed with the reconstituted drug screening gene sequence, wherein the fluorescent reporter sequence is located downstream of the precise NHEJ repair recombination screening gene sequence (i.e., downstream of the reconstituted drug screening gene sequence).

Preferably, the screening reporter gene expression cassette further comprises eukaryotic promoters and termination sequences located on either side of the transcriptional region of the expression cassette.

A system for screening for the accurate deletion of long fragment enriched cells comprising the accurate NHEJ repair screening reporter vector described above and a first targeting vector and a second targeting vector co-transfected with the screening reporter vector for the accurate deletion (e.g., by accurate NHEJ repair) of long fragments between selected loci of the genome of the cell, the long fragments having a length greater than or equal to the length of the double sgRNA target site building sequence described above.

Preferably, the long fragment is 100-1300bp.

The beneficial effects of the invention are as follows:

the invention constructs a double-target site recognition sequence structure based on an accurate NHEJ repair mechanism, and constructs a universal screening report carrier based on double sgRNA-CRISPR/Cas9 mediated accurate deletion by utilizing the sequence structure. The screening reporter vector itself can be self-cleaving and then repaired by precise NHEJ to produce the complete drug screening gene sequence. The screening report carrier and targeting carriers of various target genes are respectively co-transfected and subjected to drug screening, and then positive cell clones with the target genes with long fragments deleted accurately can be obtained through enrichment.

The invention establishes a carrier system which can be specially applied to screening and enriching long fragment accurate deleted cells based on accurate NHEJ repair by using the screening report carrier. The system can simply, conveniently and efficiently screen and enrich the precisely deleted cells on the premise of not influencing the integrity of the genome, and can ensure that the precise deletion of the gene long fragments based on precise NHEJ repair mediated by double sgRNA-CRISPR/Cas9 is applied to the research of gene functions, the cultivation of new animal varieties and the treatment of genetic diseases.

Drawings

FIG. 1 is a schematic diagram of the operation of screening the reporting vector acNHEJ-USR.

FIG. 2 is a flow chart of the operation of a transfection system combining acNHEJ-USR and targeting vector to enrich for precisely NHEJ repair mediated gene editing cells.

FIG. 3 shows the identification of the pXL-CMV-Purol (CGAagg) vector by digestion.

FIG. 4 shows the identification of the electrophoretogram of pXL-CMV-Purol (CGAagg) - (cctTCG) Puror-T2A-GFP vector by digestion.

FIG. 5 shows the identification of the map of pXL-U6-sgT 1-U6-sgT-CMV-Purol. SgT1-sgT2.Puror-T2A-GFP vector by digestion.

FIG. 6 is an acNHEJ-USR pattern.

FIG. 7 is a schematic representation of knockout in the AAVS1 site (AAVS 1 genomic locus) (acNHEJ-mediated exact deletion of a long fragment of about 1 kb).

FIG. 8 is an electrophoretogram of the cleavage assay of pX330-U6-AAVS1.SgRNA1-spCas 9.

FIG. 9 is a diagram of the restriction enzyme identification electrophoresis of pX330-U6-AAVS1.SgRNA2-spCas 9.

FIG. 10 shows the results of cell pool genomic DNA amplification.

FIG. 11 shows TA cloning results from different treatment groups.

FIG. 12 is a graph showing the effect of different screening modes on AAVS1 locus gene deletion efficiency; wherein WT%: proportion of wild-type gene copies where no deletion occurred; muNHEJ%: the proportion of gene copies deleted inaccurately; acNHEJ%: the proportion of gene copies deleted precisely.

FIG. 13 is a schematic representation of knockout in the lnc-sscg3623 locus (acNHEJ mediated exact deletion of a long fragment of about 1200 bp).

FIG. 14 shows the results of PK15 cell monoclonal sequencing analysis.

Detailed Description

The invention is described in further detail below with reference to the drawings and examples. The embodiments are presented to facilitate understanding of the technical solution of the present invention, and are not intended to limit the scope of the present invention.

Screening report vector for enriching CRISPR/Cas9 mediated precise NHEJ repair cells (Accurate NHEJ-based Universal Surrogate Reporter, acNHEJ-USR)

Referring to fig. 1, the screening report vector acNHEJ-USR constructed in the present invention includes two element units arranged in a linear relationship: one element unit mainly comprises two sgRNA expression cassettes U6-sgRNA which are all expressed by the promotion of a U6 promoter, the two sgRNA expression cassettes are mutually connected in series, and when the sgRNAs expressed by the two expression cassettes are different, the two sgRNA expression cassettes are respectively distinguished by U6-sgT1 and U6-sgT; the other element unit mainly comprises an expression cassette CMV-Purol.sgT1-sgT2.Puror-T2A-GFP which is driven by the CMV promoter and contains in the transcribed region the green fluorescent protein gene sequence GFP and the recombinant puromycin resistance gene sequence Purol.sgT1-sgT2.PruoR upstream of GFP, the GFP and Purol.sgT1-sgT2.Puror being linked by a cleavage peptide sequence (e.g.T2A). The expression cassette in both element units has a termination sequence located downstream of the transcribed region.

In the expression cassette CMV-Purol. SgT1-sgT2.Puror-T2A-GFP, the recombinant puromycin resistance gene sequence Purol. SgT1-sgT2.Puror is constructed by inserting a foreign sequence into puromycin resistance gene sequence Puro (thereby dividing Puro into two sections, namely Purol and Puror respectively), specifically inserting sequences for forming two sgRNA target sites with puromycin resistance gene target sequences located at the upper and lower positions of the cut-off position at selected cut-off positions in Puro. The puromycin resistance gene is blocked by the two sgRNA target sites, which results in the inability of the expression cassette CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP to normally express puromycin resistance and green fluorescent protein.

Since the two sgrnas target sites consist of 17bp puromycin resistance gene sequence (target sequence) and 3bp base NNN and PAM sequence NGG, respectively, the 5 'end of the exogenous sequence is NNN-NGG, the 3' end is NGG-NNN, and NNN at the 5 'end and the target sequence upstream thereof may constitute one sgRNA (e.g., sgrna.t1, sgT 1), and NNN at the 3' end and the target sequence downstream thereof may constitute another sgRNA (e.g., sgrna.t2, sgT 2).

The exogenous sequence also includes a unrelated sequence (staff sequence) that is free of the target sequence (e.g., puromycin resistance gene target sequence, cell genome target gene site target sequence described above) for separating the two target sites. The size of the irrelevant sequence is 130-150bp (literature reports that paired sgRNAs can efficiently mediate accurate deletion of genome position intervals of about 100bp, the irrelevant sequence selected by the invention is >100bp, thus not only ensuring the working efficiency of screening report carrier acNHEJ-USR in cells, namely the efficiency of reporting carrier itself to repair by accurate NHEJ, but also improving the efficiency of enriching long fragments and accurately deleting cells by using the screening report carrier).

In screening the reporter vector acNHEJ-USR, two sgRNAs transcribed from two tandem sgRNA expression cassettes (e.g., sgRNA. T1 and sgRNA. T2) can guide Cas9 to recognize and target two sgRNA target sites in the expression cassette CMV-Purol. SgT1-sgT2.Puror-T2A-GFP in the presence of Cas9, thereby triggering the repair of the break of acNHEJ-USR itself. By completing accurate NHEJ repair, the exogenous sequence is deleted, and Puro divided into two segments is accurately repaired by acNHEJ, so that an expression cassette CMV-Puro-T2A-GFP capable of normally expressing puromycin resistance and green fluorescent protein is formed.

Working principle of screening report carrier acNHEJ-USR: acNHEJ-USR itself can develop puromycin resistance by precise NHEJ repair, co-transfecting acNHEJ-USR and a cellular genome targeting vector (acNHEJ-mediated), and screening by puromycin drug, thereby enriching for gene-edited cells that undergo precise NHEJ repair (long fragment deletion).

Wherein, acNHEJ-USR can actually finish two ways of repair: (1) If acNHEJ-USR is repaired in a classical NHEJ (Classical NHEJ) mode, because random indexes are generated in the repair, puromycin resistance gene sequences cannot be accurately repaired, and the open reading frame of the genes is subjected to frame shift mutation, so that the expression cassette CMV-Purol. SgT1-sgT2.Puror-T2A-GFP still cannot be normally expressed, and cells have no puromycin resistance. (2) If acNHEJ-USR is repaired in a precise NHEJ (Accurate NHEJ) fashion, the two sgRNAs guide Cas9 to recognize the target site on Purol. SgT1-sgT2.Puror, then cleave precisely at the 3 rd base of the upstream 5 'end of the PAM sequence (the sgT sequence noted in FIG. 1 is the upstream 5' end of the PAM sequence in its complement). The resulting cleavage gap discards the middle fragment with a certain probability and is directly linked by means of precise NHEJ repair. Thus, the puromycin resistance gene is accurately repaired, the puromycin resistance gene can be expressed completely, and the cell has puromycin resistance. Meanwhile, the green fluorescent protein gene sequence GFP is serially connected behind the puromycin resistance gene sequence and is expressed together with the repair of the puromycin resistance gene. Action of green fluorescent protein: on the one hand, in order to observe the efficiency of accurate repair in the cell pool by green fluorescence, on the other hand, the drug screening of puromycin resistance genes can be replaced by a flow sorting mode.

Referring to fig. 2, the test procedure for specific screening enrichment is as follows:

1. designing a CRISPR/Cas9 targeting vector of a required editing site in a cell genome;

2. co-transfecting the constructed CRISPR/Cas9 targeting vector and acNHEJ-USR cells;

3. puromycin (puromycin) was added to the cell culture medium 48 hours after transfection;

4. screening the drug with puromycin for 3-5 days and withdrawing the drug;

5. diluting the screened cell pool by a limiting dilution method and paving the cell pool to a 96-well plate;

after 6.7 days, screening monoclonal cells, identifying by PCR, and screening positive cell monoclonal with accurate deletion;

7. positive monoclonal cells were transferred to 24-well plates and cells were expanded.

(II) screening construction example of report Carrier acNHEJ-USR

2.1 construction of pXL-CMV-Purol (CGAagg)

(1) Design of complete sgRNA-Cas9 recognition targeting sequence (target site)

Two tandem sequences with the length of 17bp are selected from puromycin resistance gene sequences to be used as target sequences of Cas 9. Then three bases and PAM sequences are artificially added to the two target sequences respectively, so that the following two target sites are obtained:

5’-ACGCCGGAGAGCGTCGACGAagg-3’

5’-cctTCGAGCGGGGGCGGTGTTCG-3’

among the above target sites, the underlined part is a target sequence selected from Puro, the italic part is added three bases, and the lowercase part is PAM sequence. The two selected target sequences are compared, have no homology with human, mouse, pig and other species, and the added three bases increase the universality of the target sequences.

(2) Designing and synthesizing a pair of primers Puro-F (BamHI) and Purol-R (XbaI) by taking a pPuro vector (any commercial vector for expressing puromycin resistance genes) as a template, and amplifying fragments BamHI-Purol (CGAagg) -XbaI (272 bp);

Puro-F(BamHI):5’-CGCggatccATGACCGAGTACAAGCCCACG-3’

PuroL-R(XbaI):5’-TGCtctagaCCTTCGTCGACGCTCTCCGGCGTGG-3’

using Takara PrimSTAR polymerase, the amplification system was as follows (applicable as follows):

2 XGC buffer:25 μL; dNTPs: 4. Mu.L; up/down primers: 1. Mu.L each (concentration 10. Mu.M); primSTAR polymerase: 0.5. Mu.L; a DNA template: 0.5 μg; water: mu.L was filled in.

Amplification procedure (applicable as follows):

1)98℃，1s；

2) 98 ℃ for 10s;62 ℃,5s;72 ℃,30s;40 cycles;

3) 72 ℃ for 10min; preserving at 10deg.C.

The fragments BamHI-Purol (CGAagg) -XbaI and pXL-BacII (Wu et al, FEBS Letters,2017,591 (6): 903-913) were digested with BamHI/XbaI double respectively (Table 1) and the digested products were recovered and ligated overnight at 16℃with T4 DNA ligase (Table 2). Coli competent DH 5. Alpha. (Tiangen Biochemical, product number CB 101) was transformed with the ligation product, screened for amp resistance, and bacterial monoclonal was selected and cultured with shaking in liquid medium at 37℃for 8 hours.

TABLE 1 cleavage reaction System

Note that: cleavage reaction conditions: enzyme digestion at 37 ℃ for 4 hours

(3) Plasmids were extracted from the cultured bacterial solutions and digested with BamHI/XbaI (Table 1). The electrophoresis bands of the positive cloning plasmid after double enzyme digestion are 4088bp and 272bp (figure 3), and the positive cloning plasmid is sequenced and verified to obtain a vector pXL-CMV-Purol (CGAagg).

TABLE 2 ligation reaction System

2.2 construction of pXL-CMV-Purol (CGAagg) - (cctTCG) Puror-T2A-GFP

(1) The pre-constructed carrier NHEJ-RPG (Dual-reporter surrogate systems for efficient enrichment of genetically modified cells) in the laboratory is used as a template, a pair of primers PuroR-F (ClaI) and polyA-R (HindIII) are designed and synthesized, and a fragment ClaI-PuroR-T2A-GFP-HindIII is amplified;

PuroR-F(ClaI):5’-CCCatcgatCCTTCGAGCGGGGGCGGTGTTCGC-3’

polyA-R(HindIII):5’-CCCaagcttTAAGATACATTGATGAGTTTGG-3’

the fragment ClaI-Puror-T2A-GFP-HindIII and the vector pXL-CMV-PuroL (CGAagg) were digested with ClaI/HindIII (Table 1), the digested products were recovered and ligated overnight at 16℃with T4 DNA ligase (Table 2). Coli competent, amp resistance screening was performed on the ligation products, bacterial monoclonal was picked and cultured with shaking in liquid medium at 37℃for 8 hours.

(2) Plasmids were extracted from the cultured broth and digested with ClaI/HindIII (Table 1). Positive plasmid clone double enzyme cutting electrophoresis band is 4343bp and1407bp (FIG. 4). The positive cloning plasmid is sequenced, and the vector pXL-CMV-Purol (CGAagg) - (cctTCG) Puror-T2A-GFP is obtained through verification. In this vector, there is 150bp of unrelated sequence (from pXL-BacII) between the fragment CGAagg and cctTCG introduced into Puro by the amplification primer, CGA and the 17bp target sequence at the 3' end of the upstream PuroLACGCCGGAGAGCGTCGA17bp target sequence constituting a sgRNA, sgT1, TCG and 5' end of PuroL downstream thereofAGCGGGGGCGGTGTTCGAnother sgRNA, sgT2, is composed, so the vector can also be referred to as pXL-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP.

2.3 construction of pXL-U6-sgT 1-U6-sgT-CMV-Purol.sgT1-sgT2.Puror-T2A-GFP

(1) Constructing two targeting vectors: pX330-U6-sgT1 and pX330-U6-sgT2

First, two pairs of fitted primers for the synthesis of sgrna.t1 (sgT 1) and sgrna.t2 (sgT 2) were designed

Puro-sgT1-F(BsaI):5’-CACCACGCCGGAGAGCGTCGACGA-3’

Puro-sgT1-R(BsaI):5’-AAACTCGTCGACGCTCTCCGGCGT-3’

Puro-sgT2-F(BsaI):5’-CACCCGAACACCGCCCCCGCTCGA-3’

Puro-sgT2-R(BsaI):5’-AAACTCGAGCGGGGGCGGTGTTCG-3’

Then, primer fitting products (the primers are diluted to 10pM, 5 mu L of each primer is absorbed and mixed uniformly) obtained through annealing are put into a PCR instrument, denatured for 10min at 95 ℃, naturally cooled to room temperature and annealed and fitted, double chains with BsaI sticky ends are obtained, namely sgRNA.T1 and sgRNA.T2, and skeleton vectors pX330-U6-Chimeric_dBSAI-CBh-hSpCas9 (Addge plasmid # 42230) used for constructing targeting vectors are inserted, so that pX330-U6-sgT1 and pX330-U6-sgT2 are respectively obtained.

(2) A pair of primers HindIII-U6-F and EcoRV-SpeI-sgT1 were designed and synthesized using pX330-U6-sgT1 as a template, and the fragment HindIII-U6-sgT1-SpeI-EcoRV was amplified;

HindIII-U6-F:5’-ccaagcttGAGGGCCTATTTCCCATGAT-3’

EcoRV-SpeI-sgT1:5’-gggatatcggactagtAGCCATTTGTCTGCAGAATT-3’

the fragments HindIII-U6-sgT1-SpeI-EcoRV and pXL-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP vectors were digested with HindIII/EcoRV respectively (Table 1), and the digested products were recovered and ligated overnight at 16℃with T4 DNA ligase (Table 2). Coli competent, amp resistance screening was performed on the ligation products, bacterial monoclonal was picked and cultured with shaking in liquid medium at 37℃for 8 hours. Plasmids were extracted from the cultured bacterial solutions and digested with BsaI/EcoRI (Table 1). The electrophoresis bands after double enzyme digestion of the positive plasmid clone are 5256bp, 3228bp and 50bp. The positive cloning plasmid is sequenced, and the vector pXL-U6-sgT1-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP is obtained through verification.

(3) Designing and synthesizing a pair of primers SpeI-U6-F and EcoRV-sgT2 by taking pX330-U6-sgT2 as a template, and amplifying a fragment SpeI-U6-sgT2-EcoRV;

SpeI-U6-F:5’-ccactagtGAGGGCCTATTTCCCATGAT-3’

EcoRV-sgT2:5’-CCgatatcCATTTGTCTGCAGAATTGGC-3’

the fragments SpeI-U6-sgT2-EcoRV and pXL-U6-sgT1-CMV-Purol. SgT1-sgT2.Puror-T2A-GFP vector were digested with SpeI/EcoRV respectively (Table 1), and the digested products were recovered and ligated overnight at 16℃with T4 DNA ligase (Table 2). Coli competent, amp resistance screening was performed on the ligation products, bacterial monoclonal was picked and cultured with shaking in liquid medium at 37℃for 8 hours. Plasmids were extracted from the cultured bacterial solutions and digested with HindIII/EcoRV (Table 1). The electrophoresis bands after double cleavage of positive plasmid clone were 6345bp and 871bp (FIG. 5). The positive cloning plasmid was sequenced and verified to give the vector pXL-U6-sgT 1-U6-sgT-CMV-Purol. SgT1-sgT2.Puror-T2A-GFP, acNHEJ-USR (FIG. 6).

(III) enrichment of precisely deleted cells Using acNHEJ-USR

3.1 human AAVS1 locus as the target gene, the AAVS1 sequence was deleted precisely long fragments by precise NHEJ (FIG. 7).

1. CRISPR/Cas9 targeting vector pX330-U6-AAVS1.SgRNA1-spCas9 for constructing AAVS1 gene

(1) Target site AAVS1-sg1: CCAGCGAGTGAAGACGGCATggg (lower case portion PAM sequence) of AAVS1 gene.

(2) Design of target site-related fitting primers according to AAVS1-sg1

AAVS1-5’target-F:5’-CACCGCCAGCGAGTGAAGACGGCAT-3’

AAVS1-5’target-R:5’-AAACATGCCGTCTTCACTCGCTGGC-3’

10 mu L (10 pM) of each synthesized fitting primer is placed in a PCR instrument and fitted through primer annealing; fitting procedure: denaturation at 94℃for 10min; and then turning off the power supply of the PCR instrument, and naturally cooling to normal temperature.

(3) The backbone vector was selected from pX330-U6-Chimeric_dBsai-CBh-hSpCas9, and the vector backbone was digested with BsaI at 37℃for 4 hours (Table 1), and the product was recovered.

(4) The digested products were ligated with T4 DNA ligase at 16℃for 6 hours (Table 2).

(5) Coli was transformed competent, amp resistance screening, bacterial plates were incubated at 37℃for 12 hours, bacterial monoclonal cells were picked from the plates, and incubated with shaking in liquid medium at 37℃for 6 hours (250 rpm).

(6) Plasmids were extracted from the cultured bacterial solutions and digested with BasI/EcoRI (Table 1). The electrophoresis band after double cleavage of the positive clone plasmid was 8509bp (FIG. 8). The positive cloning plasmid is sequenced, and the vector pX330-U6-AAVS1.sgRNA1-spCas9 (called pX330-AAVS1.sg1 for short) is obtained through verification.

2. CRISPR/Cas9 targeting vector pX330-U6-AAVS1.SgRNA2-spCas9 for constructing AAVS1 gene

(1) Target sites AAVS1-sg2: CAGGTAAAACTGACGCACGGagg (lower case portion PAM sequence) of AAVS1 gene.

(2) Target site related fitting primer designed according to AAVS1-sg2

AAVS1-3’target-F:5’-CACCGCAGGTAAAACTGACGCACGG-3’

AAVS1-3’target-R:5’-AAACCCGTGCGTCAGTTTTACCTGC-3’

(3) Specific construction steps such as connection, transformation, plasmid restriction enzyme identification (figure 9) and the like refer to the construction of the vector pX330-U6-AAVS1.SgRNA1-spCas9, and sequencing verification is carried out to obtain the vector pX330-U6-AAVS1.SgRNA2-spCas9 (called pX330-AAVS1.Sg2 for short).

Transfection treatment grouping of HEK293T cells

Non-screened group (No selection): co-transfecting cells of the paired genomic targeting vectors pX330-GOI-Cas9 (in this case pX330-AAVS1.Sg1 and pX330-AAVS1.Sg 2);

transient screening group (Transient selection): co-transfecting cells of a pair of pX330-GOI-Cas9 vector and pPuro-T2A-eGFP vector (A Universal Surrogate Reporter for Efficient Enrichment of CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells);

acNHEJ-USR screening group (acNHEJ selection): pairs of cells of the pX330-GOI-Cas9 vector and acNHEJ-USR were co-transfected.

Blank is a Blank cell (no transfection).

HEK293T cells were purchased from cell banks (catalog number: SCSP-502, shanghai)

4. Three replicates were set for each treatment group and cells from the unselected group were collected from the cell pool 48 hours after transfection. The transient screening group and the acNHEJ-USR screening group were replaced with DMEM containing 3. Mu.g/mL puromycin 48 hours after transfection, and after 3-5 days of screening, the cell survival was observed by microscopy, and when no obvious cell death was observed within 24 hours, the cell mix pool was collected.

5. Extracting genome DNA of a cell mixing pool, respectively designing PCR Detection primers Detection-F1 and Detection-R1 for synthesizing target gene loci, and amplifying corresponding sequences (figure 7);

Detection-F1:5’-CTCTGCTGTGTTGCTGCCCAAG-3’

Detection-R1:5’-CACGACCTGGTGAACACCTAGG-3’

after detecting PCR amplification products through gel electrophoresis, carrying out gray level analysis on different treatment group strips by using imageJ software, and calculating the total deletion efficiency of fragments, wherein the calculation formula is as follows:

[ deleted band gray value/(wild band gray value+deleted band gray value) ]. Times.100%.

As a result, as shown in FIG. 10, the efficiency of deletion of AAVS1 gene fragments was improved by 1.77-fold in the transient screening group (56.74%) as compared to the deletion efficiency of 32.01% in the non-screening group (No selection); deletion efficiency (94.17%) of acNHEJ-USR screening group was increased by 1.66-fold and 2.94-fold compared to the transient screening group and the no-screening group, respectively.

6. The wild type band and the knockout band were then recovered together, and after TA cloning, the selected plasmid was subjected to bacterial liquid PCR, and the gene knockout efficiency of the different treatment groups was further compared by gel electrophoresis (FIG. 11). 96 individual clones were picked for each treatment group. Wherein the gene deletion efficiency of the non-screened group was 29/96 (30.21%); the gene deletion efficiency of the transient screening group was 52/96 (54.17%); the gene deletion efficiency of the acNHEJ-USR screening group was 88/96 (91.69%). The gene deletion efficiency of the acNHEJ-USR screening group was 3.04-fold and 1.69-fold compared to the non-screening group and the transient screening group, respectively. The extracted knockout monoclonal was then sequenced and the proportion of exact deletions among all deleted clones was compared based on the sequencing results (fig. 12). Sequencing results show that the accurate deletion efficiency of the long fragment of the gene of the acNHEJ-USR screening group is 82.29%, 6.6 times of the accurate deletion efficiency of the non-screening group (12.50%) and 2.3 times of the accurate deletion efficiency of the instantaneous screening group (35.42%). Notably, the proportion of inaccurate deletions in the acNHEJ-USR screened group (9.38%) was only around 50% of the transient screened group (18.75%) or the non-screened group (17.71%).

3.2 Long fragment exact deletion of lncRNA-sscg3623 sequence by exact NHEJ was performed with the lncRNA-sscg3623 site as the target gene (FIG. 13).

1. Construction of the CRISPR/Cas9 targeting vector pX330-U6-lnc3623 sgRNA1-spCas9 of lncRNA-sscg3623

(1) The target site sg1: TTTGAAAGTTCCTCGGGCCAggg (lower case PAM sequence) of the targeting porcine lncRNA-sscg3623 gene was predicted according to on-line prediction software (http:// crispr. Mit. Edu /).

(2) Design of target site-related fitting primers based on sg1

L3623-Tar1-F:5’-CACCGTTTGAAAGTTCCTCGGGCCA-3’

L3623-Tar1-R:5’-AAACTGGCCCGAGGAACTTTCAAAC-3’

Vector construction step reference construction of CRISPR/Cas9 targeting vector of the AAVS1 gene described above.

2. Construction of the CRISPR/Cas9 targeting vector pX330-U6-lnc3623 sgRNA2-spCas9 of lncRNA-sscg3623

(1) The target site sg2: CTTCTTGAGGTCACGCACATggg (lower case PAM sequence) of the targeting porcine lncRNA-sscg3623 gene was predicted according to on-line prediction software (http:// crispr. Mit. Edu /).

(2) Design of target site-related fitting primers based on sg2

L3623-Tar2-F:5’-CACCGCTTCTTGAGGTCACGCACAT-3’

L3623-Tar2-R:5’-AAACATGTGCGTGACCTCAAGAAGC-3’

Vector construction step reference construction of CRISPR/Cas9 targeting vector of the AAVS1 gene described above.

PK15 cell transfection treatment grouping

Non-screened group (No selection): cells co-transfected with the pX330-U6-lnc3623.SgRNA1-spCas9 vector and the pX330-U6-lnc3623.SgRNA2-spCas9 vector;

acNHEJ-USR screening group (acNHEJ selection): cells co-transfected with pX330-U6-lnc3623.SgRNA1-spCas9 vector and pX330-U6-lnc3623.SgRNA2-spCas9 vector and acNHEJ-USR.

PK15 cells were purchased from ATCC (model: BH 0370)

4. Drug screening of cells was performed by adding puromycin (3. Mu.g/mL) to the acNHEJ screening group 48 hours after transfection; cells were collected 72 hours after transfection of the non-screened group. Cells were collected 5 days after drug screening in the test group.

5. Cell monoclonals were plated using limiting dilution. The collected cells were monoclonal extracted with genomic DNA, and sequences near the knockout site were amplified (see FIG. 13, F4:5'-CCCCTCATCACCGGACACAC-3'; R4: 5'-CTTTGTCCTTTTGTCAGAATCCCT-3'). And (3) after detection of the amplified product, recovering the PCR product of the knockout homozygote cell, and carrying out sequencing analysis on the recovered product.

6. As can be seen from the sequencing analysis results (FIG. 14), 30 cell monoclonals were detected in each of the unselection group and the acNHEJ-USR selection group, wherein the number of cell monoclonals in which allele long fragments were precisely deleted in the aNHEJ-USR selection group was 6, 9 heterozygous cell monoclonals were deleted, and 15 cell monoclonals were not deleted. The efficiency of long fragment deletion was 15/30 (50%), with allele deletion (deletion homozygote) efficiency of 6/30 (20%). The non-screened group did not detect single clone of cells deleted for the allele-generating fragment, deleted homozygotes were 0, and 3 single clones of heterozygote cells were deleted.

7. And (3) sequencing the PCR product of the long fragment deleted homozygous cell monoclonal, and carrying out statistical analysis on the ratio of 3/6 of the occurrence of accurate deletion of the homozygous cell monoclonal. Finally, the efficiency of the exact deletion of the biallelic gene by the cell monoclonal of the acNHEJ-USR group was 3/30 (10%).

In summary, the present invention provides a universal reporter vector that can be used exclusively for enriching double sgRNA-Cas9 mediated accurate deletion positive cells based on acNHEJ repair. The report carrier has the characteristics of self-cutting and self-repairing, the report carrier and the double sgRNA targeting carrier of the target gene are transfected into cells together, and then the cells are subjected to drug screening for 5 days, so that the cells subjected to acNHEJ mediated accurate deletion can be enriched efficiently, and the method can be suitable for accurate deletion of coding genes and non-coding genes, so that the purposes of researching gene functions and establishing gene deletion individuals are achieved. The invention successfully enriches human gene long fragment accurate deletion cells by using the established report system, and simultaneously carries out long fragment accurate deletion on long non-coding RNA of pigs, thereby verifying that the established report system not only can realize long fragment accurate deletion by using an acNHEJ repair mode, but also can improve the gene accurate deletion efficiency based on acNHEJ repair mediated by double sgRNA-Cas9, so that the invention is not only suitable for accurate deletion research of mammal genome, but also can be widely applied to gene function research and development of new animal varieties.

<110> university of agriculture and forestry science and technology in northwest

<120> screening reporter systems useful for enriching for CRISPR/Cas9 mediated precise NHEJ repair cells

<160> 29

<210>1

<211>23

<212>DNA

<213> Synthesis

<400>1

acgccggaga gcgtcgacga agg 23

<210>2

<211>23

<212>DNA

<213> Synthesis

<400>2

ccttcgagcg ggggcggtgt tcg 23

<210>3

<211>30

<212>DNA

<213> Puro-F(BamHI)

<400>3

cgcggatcca tgaccgagta caagcccacg 30

<210>4

<211>34

<212>DNA

<213> PuroL-R(XbaI)

<400>4

tgctctagac cttcgtcgac gctctccggc gtgg 34

<210>5

<211>33

<212>DNA

<213> PuroR-F(ClaI)

<400>5

cccatcgatc cttcgagcgg gggcggtgtt cgc 33

<210>6

<211>31

<212>DNA

<213> polyA-R(HindIII)

<400>6

cccaagcttt aagatacatt gatgagtttg g 31

<210>7

<211>24

<212>DNA

<213> Puro-sgT1-F(BsaI)

<400>7

caccacgccg gagagcgtcg acga 24

<210>8

<211>24

<212>DNA

<213> Puro-sgT1-R(BsaI)

<400>8

aaactcgtcg acgctctccg gcgt 24

<210>9

<211>24

<212>DNA

<213> Puro-sgT2-F(BsaI)

<400>9

cacccgaaca ccgcccccgc tcga 24

<210>10

<211>24

<212>DNA

<213> Puro-sgT2-R(BsaI)

<400>10

aaactcgagc gggggcggtg ttcg 24

<210>11

<211>28

<212>DNA

<213> HindIII-U6-F

<400>11

ccaagcttga gggcctattt cccatgat 28

<210>12

<211>36

<212>DNA

<213> EcoRV-SpeI-sgT1

<400>12

gggatatcgg actagtagcc atttgtctgc agaatt 36

<210>13

<211>28

<212>DNA

<213> SpeI-U6-F

<400>13

ccactagtga gggcctattt cccatgat 28

<210>14

<211>28

<212>DNA

<213> EcoRV-sgT2

<400>14

ccgatatcca tttgtctgca gaattggc 28

<210>15

<211>23

<212>DNA

<213> EcoRV-sgT2

<400>15

ccagcgagtg aagacggcat ggg 23

<210>16

<211>23

<212>DNA

<213> target site AAVS1-sg1 of AAVS1 Gene

<400>16

ccagcgagtg aagacggcat ggg 23

<210>17

<211>25

<212>DNA

<213> AAVS1-5’target-F

<400>17

caccgccagc gagtgaagac ggcat 25

<210>18

<211>25

<212>DNA

<213> AAVS1-5’target-R

<400>18

aaacatgccg tcttcactcg ctggc 25

<210>19

<211>23

<212>DNA

<213> target site AAVS1-sg2 of AAVS1 Gene

<400>19

caggtaaaac tgacgcacgg agg 23

<210>20

<211>25

<212>DNA

<213> AAVS1-3’target-F

<400>20

caccgcaggt aaaactgacg cacgg 25

<210>21

<211>25

<212>DNA

<213> AAVS1-3’target-F

<400>21

aaacccgtgc gtcagtttta cctgc 25

<210>22

<211>22

<212>DNA

<213> Detection-F1

<400>22

ctctgctgtg ttgctgccca ag 22

<210>23

<211>22

<212>DNA

<213> Detection-R1

<400>23

cacgacctgg tgaacaccta gg 22

<210>24

<211>25

<212>DNA

<213> L3623-Tar1-F

<400>24

caccgtttga aagttcctcg ggcca 25

<210>25

<211>25

<212>DNA

<213> L3623-Tar1-R

<400>25

aaactggccc gaggaacttt caaac 25

<210>26

<211>25

<212>DNA

<213> L3623-Tar2-F

<400>26

caccgcttct tgaggtcacg cacat 25

<210>27

<211>25

<212>DNA

<213> L3623-Tar2-R

<400>27

aaacatgtgc gtgacctcaa gaagc 25

<210>28

<211>20

<212>DNA

<213> F4

<400>28

cccctcatca ccggacacac 20

<210>29

<211>24

<212>DNA

<213> R4

<400>29

ctttgtcctt ttgtcagaat ccct 24

Claims (6)

1. An accurate NHEJ repair screening report vector, characterized by: the gene expression cassette comprises a screening reporter gene expression cassette and an sgRNA expression cassette, wherein a transcription region of the screening reporter gene expression cassette comprises an accurate NHEJ repair recombination screening gene sequence; the accurate NHEJ repair recombination screening gene sequence comprises a double sgRNA target site construction sequence for inserting into a drug screening gene sequence blocking position in the transcription region, wherein the double sgRNA target site construction sequence comprises a three-base sequence for forming two sgRNA sequences with the drug screening gene target sequences positioned at the upstream and downstream of the blocking position and two PAM sequences respectively connected with the corresponding sgRNA sequences, and the double sgRNA target site respectively uses the sgRNA transcribed by the sgRNA expression cassette as a guide sequence for targeting the accurate NHEJ repair recombination screening gene sequence;

the double sgRNA target site building sequence further includes an unrelated sequence for linking the two PAM sequences; the precise NHEJ repair screening report vector carries out precise NHEJ repair by breaking CRISPR/Cas9 mediated double sgRNA target sites, and restores drug screening gene sequences in the transcription region of the screening report gene expression cassette and expresses corresponding drug resistance.

2. The precise NHEJ repair screening report vector of claim 1, wherein: the transcribed region of the screening reporter gene expression cassette further comprises a fluorescent reporter gene sequence, the fluorescent reporter gene sequence is co-expressed with the drug screening gene sequence, and the fluorescent reporter gene sequence is located downstream of the drug screening gene sequence.

3. A precision NHEJ repair screening report vector according to claim 1 or 2, characterized in that: the screening reporter gene expression cassette further comprises a promoter and a termination sequence flanking the transcribed region.

4. A system for screening for enriched long fragment precise deletion of cells, characterized by: a targeting vector comprising an accurate NHEJ repair screening reporter vector and a long fragment co-transfected with the screening reporter vector into a target editing cell for accurate deletion of selected loci within the genome of the cell; the precise NHEJ repair screening report vector comprises a screening report gene expression cassette and an sgRNA expression cassette, and a transcription region of the screening report gene expression cassette comprises a precise NHEJ repair recombination screening gene sequence; the accurate NHEJ repair recombination screening gene sequence comprises a double sgRNA target site construction sequence for inserting into a drug screening gene sequence blocking position in the transcription region, wherein the double sgRNA target site construction sequence comprises a three-base sequence for forming two sgRNA sequences with the drug screening gene target sequences positioned at the upstream and downstream of the blocking position and two PAM sequences respectively connected with the corresponding sgRNA sequences, and the double sgRNA target site respectively uses the sgRNA transcribed by the sgRNA expression cassette as a guide sequence for targeting the accurate NHEJ repair recombination screening gene sequence; the length of the long fragment is greater than or equal to that of the double sgRNA target site construction sequence;

the double sgRNA target site building sequence further includes an unrelated sequence for linking the two PAM sequences; the precise NHEJ repair screening report vector carries out precise NHEJ repair by breaking CRISPR/Cas9 mediated double sgRNA target sites, and restores drug screening gene sequences in the transcription region of the screening report gene expression cassette and expresses corresponding drug resistance.

5. The system for screening for accurate deletion of long fragment enriched cells as set forth in claim 4, wherein: the long fragment is 100-1300bp.

6. Use of the precise NHEJ repair screen reporter vector of claim 1 in enriching for exactly deleted gene knockout cells of a long fragment of a biallelic gene.

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