CN111876422A - Screening report system capable of being used for enriching CRISPR/Cas9-mediated accurate NHEJ repair cells - Google Patents

Abstract

The invention discloses a screening reporter system that can be used for enriching CRISPR/Cas9-mediated precise NHEJ repair cells, including an accurate NHEJ repair screening reporter vector and a long cell genome fragment co-transfected with the screening reporter vector in target editing cells Targeting vector, accurate NHEJ repair The screening reporter vector includes a screening reporter gene expression cassette and two sgRNA expression cassettes, and the transcription region of the screening reporter gene expression cassette includes a double sgRNA target site for insertion at the selected interrupted position of the drug screening gene sequence The sgRNAs transcribed by the two sgRNA expression cassettes were used as the guide sequences for targeting and screening the reporter gene expression cassettes at the double sgRNA target sites. The screening reporter system of the present invention can efficiently enrich positive cell clones with precise deletion of long fragments of target genes by co-transfection and drug screening.

Description

A screening report that can be used to enrich cells for CRISPR/Cas9-mediated precise NHEJ repair reporting system

technical field

The invention belongs to the technical field of genetic engineering, and relates to a construction method of a screening reporter vector that can be used to enrich CRISPR/Cas9-mediated precise NHEJ repair cells and its application in precise deletion of long fragments of cell genomes.

Background technique

Precise editing can safely modify the genome, and is becoming an important means of studying gene function and breeding new animal breeds. CRISPR/Cas9 technology can modify the target gene more easily and efficiently. In the CRISPR/Cas9 system, Cas9 nuclease, under the guidance of single-stranded guide RNA (sgRNA), can target and recognize the target genomic locus and induce DNA of double-strand breaks (DSBs). There are two main ways of DNA double-strand break repair (Repair): non-homologous end joining (NHEJ) and homologous recombination (HDR). Classical NHEJ is considered an error-prone DSB repair pathway, often resulting in random insertions or deletions (indels) at target sites. HDR is more suitable for precise editing such as single base substitution, target fragment knock-in and knock-out.

The classical NHEJ pathway-mediated gene fragment deletion has always been the preferred method for gene deletion, but due to the characteristics of small indels, the deleted genotype cannot be accurately predicted and set. Therefore, individuals with classical NHEJ-mediated gene deletions have some degree of genetic safety concerns. Although HDR-mediated donor-dependent gene fragment deletion can achieve the purpose of precise editing, the efficiency of HDR repair is much lower than that of NHEJ repair in practical applications, and the repair efficiency of HDR increases with the length of the edited fragment (donor integration). Prolonged and reduced, it is difficult to widely apply.

In recent years, studies have found a DNA double-strand break repair method different from the classical NHEJ pathway. When Cas9 nuclease causes DNA double-strand breaks, the free ends of DNA are not digested and modified, but are replaced by DNA. Ligase directly joins the broken nicks. This repair method is called accurate NHEJ repair (Accurate-NHEJ, acNHEJ for short). The repair of acNHEJ is precise and predictable, and the repair efficiency of acNHEJ is almost the same as that of classical NHEJ. However, the length of acNHEJ repair-mediated precise fragment deletion is only within 100 bp, and there is no report on acNHEJ-mediated precise deletion of long fragments.

The combination of CRISPR/Cas9 and NHEJ-mediated DSBs repair is commonly used in gene deletion studies. However, with the continuous innovation of gene editing technology, people's research and application of gene editing on individual animals has begun to change to a safe and precise direction. Therefore, whether the target gene can be edited efficiently and accurately has become the standard for whether gene editing tools can be widely used. At present, the technical problem to be solved urgently is to improve the efficiency of precise deletion of long gene fragments and to develop a corresponding gene editing/screening reporting system.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a screening reporter system that can be used to enrich CRISPR/Cas9-mediated precise NHEJ repair cells.

To achieve the above object, the present invention has adopted the following technical solutions:

An accurate NHEJ repair recombination screening gene, comprising a dual sgRNA target site construction sequence for insertion at a screening/reporter gene (e.g., drug screening gene) sequence at a selected interrupter position (interrupter positions are located at two of the drug screening gene sequences) Between the bases, the drug screening gene is recombined by inserting the double sgRNA target site construction sequence, and the expression of the drug screening gene can be blocked), and the double sgRNA target site construction sequence includes a structure for and is located in the blocking position. The upstream and downstream gene (eg, drug screening genes) target sequences constitute the three-base sequence of the two sgRNA sequences and the two PAM sequences respectively linked to the corresponding sgRNA sequences.

Preferably, the double sgRNA target site construction sequence also includes an unrelated sequence (without any target sequence) for connecting the two PAM sequences.

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

An accurate NHEJ repair screening reporter vector, including a screening reporter gene expression cassette and two sgRNA expression cassettes, the transcription region of the screening reporter gene expression cassette includes a fluorescent reporter gene sequence and a drug screening gene (and the above-mentioned dual sgRNA target site) Construction sequence) the precise NHEJ repair recombination screening gene sequence constructed, the dual sgRNA target sites respectively use the sgRNA transcribed by the two sgRNA expression cassettes as the guide sequence for targeting the precise NHEJ repair recombination screening gene sequence.

Preferably, the precise NHEJ repair screening reporter vector, for example, in the transfected target editing cells, by CRISPR/Cas9-mediated reporter vector itself (specifically refers to the precise NHEJ repair recombination screening gene sequence) dual sgRNA target site The break is subjected to precise NHEJ repair, and the transcriptional region of the screening reporter gene expression cassette is restored (ie, the partition is eliminated by precise deletion of the double sgRNA target site construction sequence), and the drug screening gene sequence is obtained and the precise NHEJ repair screening reporter vector can express the corresponding drug resistance.

Preferably, the fluorescent reporter gene sequence and the restored drug screening gene sequence are co-expressed, wherein the fluorescent reporter gene sequence is located downstream of the precise NHEJ repair recombination screening gene sequence (ie, downstream of the restored drug screening gene sequence).

Preferably, the screening reporter gene expression cassette further comprises eukaryotic promoter and termination sequences located on both sides of the transcription region of the expression cassette.

A system for screening cells enriched for precise deletion of long fragments, comprising the above-mentioned precise NHEJ repair screening reporter vector and co-transfected with the screening reporter vector in a target edited cell for precise deletion (eg, by precise NHEJ repair) of the genome of the cell The first targeting vector and the second targeting vector of the long fragment between the selected sites, the length of the long fragment is greater than or equal to the length of the above-mentioned double sgRNA target site construction sequence.

Preferably, the long fragment is 100-1300 bp.

The beneficial effects of the present invention are embodied in:

Based on the precise NHEJ repair mechanism, the invention constructs a double target site recognition sequence structure, and uses the sequence structure to construct a universal screening reporter carrier based on double sgRNA-CRISPR/Cas9-mediated precise deletion. The screening reporter vector itself can be self-cleaving and then repaired by precise NHEJ to generate a complete drug screening gene sequence. By co-transfecting the screening reporter vector with a variety of different target gene targeting vectors and performing drug screening, positive cell clones with precise deletion of long fragments of the target gene can be enriched.

The present invention utilizes the above-mentioned screening reporter carrier to establish a carrier system that can be specially used for screening and enriching cells with precise deletion of long fragments based on precise NHEJ repair. Under the premise of not affecting the integrity of the genome, the system can be easily and efficiently screened and enriched for precisely deleted cells, and can accurately delete long gene fragments based on precise NHEJ repair mediated by dual sgRNA-CRISPR/Cas9 It is used in the research of gene function, the breeding of new animal breeds and the treatment of genetic diseases.

Description of drawings

Figure 1 is a schematic diagram of the work of screening the reporter vector acNHEJ-USR.

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

Figure 3 is an electrophoresis image of pXL-CMV-PuroL (CGAagg) vector digestion and identification.

Figure 4 is an electrophoresis image of pXL-CMV-PuroL(CGAagg)-(cctTCG)PuroR-T2A-GFP vector digestion and identification.

Fig. 5 is an electrophoresis image of pXL-U6-sgT1-U6-sgT2-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP vector digestion and identification.

Figure 6 is an acNHEJ-USR map.

Figure 7 is a schematic diagram of the knockout in the AAVS1 locus (AAVS1 genomic locus) (acNHEJ-mediated precise deletion of ~1 kb long fragments).

Figure 8 is an electrophoresis image of the restriction enzyme digestion identification of pX330-U6-AAVS1.sgRNA1-spCas9.

Fig. 9 is an electrophoresis image of restriction enzyme digestion identification of pX330-U6-AAVS1.sgRNA2-spCas9.

Figure 10 shows the results of genomic DNA amplification in the cell pool.

Figure 11 shows the results of TA cloning in different treatment groups.

Figure 12 shows the effect of different screening methods on the gene deletion efficiency of AAVS1 locus; among them, WT%: the proportion of wild-type gene copies that have not been deleted; muNHEJ%: the proportion of non-exactly deleted gene copies; acNHEJ%: exact deletion The proportion of gene copies.

Figure 13 is a schematic diagram of the knockout in the lnc-sscg3623 site (acNHEJ-mediated precise deletion of a long fragment of about 1200 bp).

Figure 14 shows the results of single-clonal sequencing analysis of PK15 cells.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are to make the technical solutions of the present invention easier to understand, rather than to limit the protection scope of the present invention.

(1) A screening reporter vector (AccurateNHEJ-based Universal Surrogate Reporter, acNHEJ-USR) for enriching CRISPR/Cas9-mediated precise NHEJ repair cells

Referring to Fig. 1, the screening reporter vector acNHEJ-USR constructed by the present invention includes two element units arranged in a linear relationship: one of the element units mainly includes two sgRNA expression cassettes U6-sgRNA, both of which are expressed by the U6 promoter. The two sgRNA expression cassettes are connected in series. When their respective sgRNAs are different, the two sgRNA expression cassettes are distinguished by U6-sgT1 and U6-sgT2 respectively; the other element unit mainly includes the expression promoted by the CMV promoter, and contains The expression cassettes CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP, GFP and PuroL.sgT1-sgT2 .PuroR is linked by a cleavage peptide sequence (eg, T2A). The expression cassettes in both element units have termination sequences 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 an exogenous sequence inserted into the puromycin resistance gene sequence Puro And the constructed (thus dividing Puro into two sections, denoted as PuroL and PuroR respectively), specifically, inserting the puromycin resistance gene target sequence at the selected blocking position in Puro for connecting with the puromycin resistance gene target sequence located upstream and downstream of the blocking position Sequences that make up the two sgRNA target sites. The puromycin resistance gene was interrupted by these two sgRNA target sites, resulting in the inability of the expression cassette CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP to express puromycin resistance and green fluorescent protein normally.

Since the two sgRNA target sites are composed of a 17bp puromycin resistance gene sequence (target sequence), a 3bp base NNN and a PAM sequence NGG, the 5' end of the above exogenous sequence is NNN-NGG, 3 The 'end is NGG-NNN, and the NNN at the 5' end and its upstream target sequence can form one sgRNA (for example, sgRNA.T1, sgT1 for short), and the NNN at the 3' end and its downstream target sequence can form another sgRNA (for example, sgRNA .T2, referred to as sgT2).

The exogenous sequence also includes a staff sequence that does not contain a target sequence (eg, the above-mentioned puromycin resistance gene target sequence, cellular genome target gene site target sequence) for separating the two target sites. ). The size of the irrelevant sequence is 130-150bp (it is reported in the literature that paired sgRNA can efficiently mediate the precise deletion of the genome position interval of about 100bp, and the irrelevant sequence selected in the present invention is >100bp, which not only ensures that the screening reporter vector acNHEJ-USR is in the cell The efficiency of the work, that is, the efficiency of the reporter vector itself through accurate NHEJ repair, and at the same time improve the efficiency of using the screening reporter vector to enrich long fragments and accurately delete cells).

In the screening reporter vector acNHEJ-USR, two sgRNAs (eg, sgRNA.T1 and sgRNA.T2) transcribed from two tandem sgRNA expression cassettes can guide Cas9 to recognize and target the expression cassette CMV in the presence of Cas9 - Two sgRNA target sites in PuroL.sgT1-sgT2.PuroR-T2A-GFP, thereby triggering break repair in acNHEJ-USR itself. By completing accurate NHEJ repair, the exogenous sequence is deleted, and the Puro separated into two segments is accurately repaired by acNHEJ to form an expression cassette CMV-Puro-T2A-GFP that can normally express puromycin resistance and green fluorescent protein.

The working principle of the screening reporter vector acNHEJ-USR: acNHEJ-USR itself can generate puromycin resistance through precise NHEJ repair, co-transfection of acNHEJ-USR and the cellular genome targeting vector (acNHEJ-mediated), and through the puromycin drug Screening to enrich for gene-edited cells that undergo precise NHEJ repair (long fragment deletion).

Among them, acNHEJ-USR can actually be repaired in two ways: (1) If acNHEJ-USR is repaired in the classical NHEJ (Classical NHEJ) way, since this repair will generate random indels, making puromycin resistant The gene sequence cannot be accurately repaired, resulting in a frameshift mutation in the open reading frame of the gene. Therefore, the expression cassette CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP still cannot be expressed normally, and the cells do not have puromycin resistance. (2) If acNHEJ-USR is repaired in an Accurate NHEJ (Accurate NHEJ) manner, the two sgRNAs guide Cas9 to recognize the target site on PuroL.sgT1-sgT2.PuroR, and then at the upstream 5' end of the PAM sequence (labeled in Figure 1) The sgT2 sequence is cleaved at the 3rd base at the upstream 5' end of the PAM sequence in its complementary sequence, respectively. The resulting break gaps lose the intermediate fragments with a certain probability, and are directly connected by precise NHEJ repair. In this way, the puromycin resistance gene can be accurately repaired and fully expressed, and the cells are puromycin resistant. At the same time, the green fluorescent protein gene sequence GFP is connected in series behind the puromycin resistance gene sequence, and is expressed together with the repair of the puromycin resistance gene. The role of green fluorescent protein: on the one hand, it is to observe the efficiency of precise repair in the cell pool through green fluorescence, and on the other hand, it can also replace the drug screening of puromycin resistance gene by flow sorting.

Referring to Figure 2, the specific experimental process of screening and enrichment is as follows:

1. Design a CRISPR/Cas9 targeting vector that requires editing sites in the cell genome;

2. Co-transfect cells with the constructed CRISPR/Cas9 targeting vector and acNHEJ-USR;

3. 48 hours after transfection, add puromycin to the cell culture medium;

4. Withdrawal after 3 to 5 days of screening with puromycin;

5. Dilute the screened cell pool by limiting dilution and spread it to a 96-well plate;

After 6.7 days, screen monoclonal cells, identify them by PCR, and screen the positive cell monoclones that are accurately deleted;

7. The positive monoclonal cells were transferred to a 24-well plate, and the cells were expanded.

(2) Construction example of screening reporter vector acNHEJ-USR

2.1 Construction of pXL-CMV-PuroL (CGAagg)

(1) Design a complete sgRNA-Cas9 to recognize the target sequence (target site)

In the puromycin resistance gene sequence, two tandem sequences with a length of 17 bp were selected as the target sequence of Cas9. Then, artificially add three bases and PAM sequences to the two target sequences to obtain the following two target sites:

5'- ACGCCGGAGAGCGTCGA CGAagg-3'

5'-cctTCG AGCGGGGGCGGTGTTCG -3'

In the above target sites, the underlined part is the target sequence selected from Puro, the italicized part is the added three bases, and the lowercase part is the PAM sequence. The two selected target sequences have no homology with human, mouse, pig and other species after alignment, and the added three bases increase the versatility of the target sequence.

(2) Design and synthesize a pair of primers Puro-F (BamHI) and PuroL-R (XbaI) with pPuro vector (any commercial vector expressing puromycin resistance gene can be used) as a template to amplify the fragment BamHI -PuroL(CGAagg)-XbaI(272bp);

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

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

Using Takara PrimSTAR polymerase, the amplification system is as follows (all of the following apply):

2×GC buffer: 25 μL; dNTPs: 4 μL; upstream/downstream primers: 1 μL each (concentration 10 μM); PrimSTAR polymerase: 0.5 μL; DNA template: 0.5 μg; water: make up 50 μL.

Amplification procedure (all of the following apply):

1) 98℃, 1s;

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

3) 72℃, 10min; 10℃, save.

Fragments BamHI-PuroL(CGAagg)-XbaI and pXL-BacII (Wu et al., FEBS Letters, 2017, 591(6): 903-913) were digested with BamHI/XbaI double enzymes (Table 1), and the products were digested After recovery, they were ligated with T4 DNA ligase overnight at 16°C (Table 2). The ligation product was transformed into E. coli competent DH5α (Tiangen Biochemical, product number CB101), and amp resistance was screened. Bacterial single clones were picked and cultured in liquid medium at 37°C for 8 hours with shaking.

Table 1. Enzymatic digestion reaction system

Note: Reaction conditions for digestion: 37°C for 4 hours

(3) The plasmid was extracted from the cultured bacterial liquid and digested with BamHI/XbaI double enzymes (Table 1). The electrophoresis bands of the positive cloned plasmid were 4088bp and 272bp after double-enzyme digestion (Figure 3). The positive cloned plasmid was sequenced to verify that the vector pXL-CMV-PuroL (CGAagg) was obtained.

Table 2. Ligation reaction system

2.2 Construction of pXL-CMV-PuroL(CGAagg)-(cctTCG)PuroR-T2A-GFP

(1) Using the vector NHEJ-RPG (Dual-reporter surrogate systems for efficient enrichment of genetically modified cells) constructed in our laboratory as a template, a pair of primers PuroR-F(ClaI) and polyA-R(HindIII) were designed and synthesized. Amplified fragment ClaI-PuroR-T2A-GFP-HindIII;

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 double enzymes respectively (Table 1), and the digested products were recovered and ligated with T4 DNA ligase at 16°C overnight (Table 1). 2). The ligation product was transformed into E. coli competent, and the amp resistance was screened. Bacterial monoclones were picked and cultured in liquid medium at 37° C. for 8 hours with shaking.

(2) The plasmid was extracted from the cultured bacterial liquid and digested with ClaI/HindIII double enzyme (Table 1). The electrophoresis bands of the positive plasmid clones after double digestion were 4343bp and 1407bp (Fig. 4). The positive cloned plasmid was sequenced and verified to obtain the vector pXL-CMV-PuroL(CGAagg)-(cctTCG)PuroR-T2A-GFP. In this vector, there is a 150bp irrelevant sequence (from pXL-BacII) between the fragment CGAagg and cctTCG introduced into Puro by the amplification primer. CGA and the 17bp target sequence ACGCCGGAGAGCGTCGA at the 3' end of the upstream PuroL form a sgRNA, namely sgT1, TCG The 17bp target sequence AGCGGGGCGGTGTTCG at the 5' end of its downstream PuroL forms another sgRNA, namely sgT2, so this vector can also be called pXL-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP.

2.3 Construction of pXL-U6-sgT1-U6-sgT2-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP

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

First, two pairs of fitting primers for the synthesis of sgRNA.T1 (sgT1) and sgRNA.T2 (sgT2) 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'-AAAACTCGAGCGGGGGCGGTGTTCG-3'

Then, the primer fitting products obtained by annealing (primers were diluted to 10 pM, 5 μL of each was mixed, placed in a PCR machine, denatured at 95°C for 10 min, naturally cooled to room temperature for annealing and fitting, and double-stranded with BsaI sticky ends was obtained. ), namely sgRNA.T1 and sgRNA.T2, were inserted into the backbone vector pX330-U6-Chimeric_dBsaI-CBh-hSpCas9 (Addgene plasmid#42230) used to construct the targeting vector, resulting in pX330-U6-sgT1 and pX330-U6-sgT2, respectively.

(2) Using pX330-U6-sgT1 as a template, design and synthesize a pair of primers HindIII-U6-F and EcoRV-SpeI-sgT1 to amplify the fragment HindIII-U6-sgT1-SpeI-EcoRV;

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 double enzymes (Table 1), and the digested products were recovered and ligated with T4 DNA Enzymes were ligated overnight at 16°C (Table 2). The ligation product was transformed into E. coli competent, and the amp resistance was screened. Bacterial monoclones were picked and cultured in liquid medium at 37° C. for 8 hours with shaking. The plasmid was extracted from the cultured bacterial liquid and digested with BsaI/EcoRI (Table 1). The electrophoresis bands of the positive plasmid clones after double digestion were 5256bp, 3228bp and 50bp. The positive cloned plasmid was sequenced and verified to obtain the vector pXL-U6-sgT1-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP.

(3) Using pX330-U6-sgT2 as a template, design and synthesize a pair of primers SpeI-U6-F and EcoRV-sgT2 to amplify the fragment SpeI-U6-sgT2-EcoRV;

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

EcoRV-sgT2:5'-CCgatatcCATTTGTCTGCAGAGAATTGGC-3'

The fragment SpeI-U6-sgT2-EcoRV and pXL-U6-sgT1-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP vector were digested with SpeI/EcoRV double enzyme (Table 1), and the digested product was recovered with T4 DNA ligase was ligated overnight at 16°C (see Table 2). The ligation product was transformed into E. coli competent, and the amp resistance was screened. Bacterial monoclones were picked and cultured in liquid medium at 37° C. for 8 hours with shaking. Plasmids were extracted from the cultured bacterial liquid and digested with HindIII/EcoRV double enzymes (Table 1). The electrophoresis bands of the positive plasmid clones after double digestion were 6345bp and 871bp (Fig. 5). The positive cloned plasmid was sequenced and verified to obtain the vector pXL-U6-sgT1-U6-sgT2-CMV-PuroL.sgT1-sgT2.PuroR-T2A-GFP, namely acNHEJ-USR (Fig. 6).

(3) Using acNHEJ-USR to enrich precisely deleted cells

3.1 Using the human AAVS1 locus as the target gene, the long fragment of the AAVS1 sequence was precisely deleted by precise NHEJ (Fig. 7).

1. Construction of CRISPR/Cas9 targeting vector pX330-U6-AAVS1.sgRNA1-spCas9 for AAVS1 gene

(1) The target site of AAVS1 gene AAVS1-sg1: CCAGCGAGTGAAGACGGCATggg (the lowercase part is PAM sequence).

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

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

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

Place 10 μL (10 pM) of the synthesized fitting primers in a PCR machine, and fit by primer annealing; fitting program: denaturation at 94°C for 10 minutes; then turn off the power of the PCR machine and naturally cool to room temperature.

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

(4) The digested product was ligated with T4 DNA ligase at 16°C for 6 hours (Table 2).

(5) Transformed E. coli competent, amp resistance screening, bacterial plates were cultured at 37° C. for 12 hours, and bacterial monoclones in the plate were picked and cultured in liquid medium with shaking at 37° C. for 6 hours (250 rpm).

(6) The plasmid was extracted from the cultured bacterial liquid and digested with BasI/EcoRI double enzyme (Table 1). The electrophoresis band of the positive clone plasmid was 8509bp after double digestion (Fig. 8). The positive cloned plasmid was sequenced and verified to obtain the vector pX330-U6-AAVS1.sgRNA1-spCas9 (pX330-AAVS1.sg1 for short).

2. Construction of CRISPR/Cas9 targeting vector pX330-U6-AAVS1.sgRNA2-spCas9 for AAVS1 gene

(1) The target site of AAVS1 gene AAVS1-sg2: CAGGTAAAACTGACGCACGGagg (the lowercase part is PAM sequence).

(2) Design target site-related fitting primers according to AAVS1-sg2

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

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

(3) The specific construction steps such as ligation, transformation, and identification of plasmid digestion (Fig. 9) refer to the construction of the above vector pX330-U6-AAVS1.sgRNA1-spCas9, and the sequencing verification obtains the vector pX330-U6-AAVS1.sgRNA2-spCas9 (referred to as pX330-AAVS1.sg2).

3. Transfection treatment grouping of HEK293T cells

No selection group: cells co-transfected with paired genome targeting vectors pX330-GOI-Cas9 (pX330-AAVS1.sg1 and pX330-AAVS1.sg2 in this case);

Transient selection: cells co-transfected with paired 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 selection group (acNHEJ selection): cells co-transfected with paired pX330-GOI-Cas9 vector and acNHEJ-USR.

Blank is blank cells (no transfection).

HEK293T cells were purchased from Cell Bank (catalog number: SCSP-502, Shanghai )

4. Three replicates were set for each treatment group, and the cells of the unscreened group were collected in a mixed pool of cells 48 hours after transfection. The transient screening group and the acNHEJ-USR screening group were replaced with DMEM containing 3 μg/mL puromycin 48 hours after transfection. After 3 to 5 days of screening, the cell survival was observed by microscope, and there was no obvious cell death within 24 hours. , collect the cells in the mixed pool.

5. Extract genomic DNA from the mixed pool of cells, design and synthesize PCR detection primers Detection-F1 and Detection-R1 of the target gene loci respectively, and amplify the corresponding sequences (Figure 7);

Detection-F1: 5'-CTCTGCTGTGTTGCTGCCCAAG-3'

Detection-R1: 5'-CACGACCTGGTGAACACCTAGG-3'

After the PCR amplification products were detected by gel electrophoresis, the grayscale analysis of the bands in different treatment groups was performed using ImageJ software, and the total deletion efficiency of the fragments was calculated. The calculation formula is:

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

The results are shown in Figure 10. Compared with the 32.01% deletion efficiency of the unselected group (No selection), the deletion efficiency of the AAVS1 gene fragment in the transient selection group (56.74%) was increased by 1.77 times; the deletion efficiency of the acNHEJ-USR selection group ( 94.17%) were 1.66 times and 2.94 times higher than those of the transient screening group and the non-screening group, respectively.

6. The wild-type band and the knockout band are then recovered by gel together. After being cloned by TA, the single clone of the picked plasmid is subjected to bacterial liquid PCR, and the gene knockout efficiency of different treatment groups is further compared by gel electrophoresis (Fig. 11). 96 single clones were picked from each treatment group. The gene deletion efficiency of the unscreened group was 29/96 (30.21%); the gene deletion efficiency of the transient selection group was 52/96 (54.17%); the gene deletion efficiency of the acNHEJ-USR selection group was 88/96 (91.69%) . The gene deletion efficiency of the acNHEJ-USR screening group was 3.04 times and 1.69 times that of the unscreened group and the transient screening group, respectively. The extracted knockout monoclones were then sequenced, and the exact deletion ratios among all deleted clones were compared according to the sequencing results (Fig. 12). The sequencing results showed that the precise deletion efficiency of long gene fragments in the acNHEJ-USR screening group was 82.29%, which was 6.6 times that of the unscreened group (12.50%) and 2.3 times that of the transient screening group (35.42%). It is worth noting that the proportion of imprecise deletions in the acNHEJ-USR screening group (9.38%) was only about 50% of the transient screening group (18.75%) or the unscreened group (17.71%).

3.2 Using the lncRNA-sscg3623 locus as the target gene, the long fragment of the lncRNA-sscg3623 sequence was accurately deleted by precise NHEJ (Figure 13).

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

(1) According to the online prediction software (http://crispr.mit.edu/), the target site sg1:TTTGAAAGTTCCTCGGGCCAggg (the lowercase part is the PAM sequence) was predicted to target the pig lncRNA-sscg3623 gene.

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

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

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

The vector construction steps refer to the construction of the above-mentioned CRISPR/Cas9 targeting vector of AAVS1 gene.

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

(1) According to the online prediction software (http://crispr.mit.edu/), the target site sg2: CTTCTTGAGGTCACGCACATggg (the lowercase part is the PAM sequence) was predicted to target the pig lncRNA-sscg3623 gene.

(2) Design target site-related fitting primers according to sg2

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

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

The vector construction steps refer to the construction of the above-mentioned CRISPR/Cas9 targeting vector of AAVS1 gene.

3. PK15 cell transfection treatment grouping

No selection group: cells co-transfected with pX330-U6-lnc3623.sgRNA1-spCas9 vector and pX330-U6-lnc3623.sgRNA2-spCas9 vector;

acNHEJ-USR selection 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: BH0370)

4. After 48 hours of transfection, puromycin (3 μg/mL) was added to the acNHEJ screening group for drug screening; the cells of the unscreened group were collected 72 hours after transfection. The cells of the experimental group were collected after 5 days of drug screening.

5. Plate cell clones by limiting dilution method. Cell genomic DNA was extracted from the collected cell monoclonal, and the sequence near the knockout site was amplified (refer to Figure 13, F4: 5'-CCCCTCATCACCGGACACAC-3'; R4: 5'-CTTTGTCCTTTTGTCAGAATCCCT-3'). After detection of amplification products, the PCR products of knockout homozygous cells were recovered, and the recovered products were subjected to sequencing analysis.

6. The results of sequencing analysis can be seen (Figure 14), the unscreened group and the acNHEJ-USR screening group each detected 30 cell monoclones, of which the aNHEJ-USR screening group had precise deletion of long allele fragments. The number was 6, 9 were deleted heterozygous cells, and 15 were not deleted. The efficiency of long fragment deletion was 15/30 (50%), and the efficiency of allelic deletion (deletion homozygotes) was 6/30 (20%). In the unscreened group, no single clones of cells with long fragment deletion of alleles were detected, 0 homozygotes for deletion, and 3 single clones for deletion heterozygotes.

7. The PCR product of the homozygous cell monoclonal with long fragment deletion is sent to sequencing, and the proportion of exact deletion of the homozygous cell monoclonal deleted by statistical analysis is 3/6. Finally, the acNHEJ-USR group of cells had a 3/30 (10%) efficiency of precise biallelic deletion in monoclonal cells.

In conclusion, the present invention provides a universal reporter vector that can be specifically used to enrich double sgRNA-Cas9-mediated acNHEJ repair-based precise deletion-positive cells. The reporter vector has the characteristics of self-cleavage and self-repair. The reporter vector and the double sgRNA targeting vector targeting the target gene are co-transfected into the cells, and after 5 days of drug screening, the cells can be efficiently enriched and acNHEJ-mediated The induced precise deletion of cells can be applied to the precise deletion of coding genes and non-coding genes, so as to achieve the purpose of studying gene function and establishing gene deletion individuals. Using the established reporter system, the present invention successfully enriches the cells with precise deletion of human gene long fragments, and at the same time, accurately deletes long fragments of porcine long-chain non-coding RNAs, thereby verifying that the established reporter system can not only use The acNHEJ repair method realizes precise deletion of long fragments, and at the same time can improve the efficiency of precise deletion of genes based on acNHEJ repair mediated by dual sgRNA-Cas9. Therefore, the present invention is not only suitable for precise deletion research on mammalian genomes, but also can be more widely used in Gene function research and development of new animal species.

<110> Northwest A&F University

<120> A screening reporter system that can be used to enrich cells for CRISPR/Cas9-mediated precise NHEJ repair

<160> 29

<210>1

<211>23

<212> DNA

<213> Synthetic

<400>1

acgccggaga gcgtcgacga agg 23

<210>2

<211>23

<212> DNA

<213> Synthetic

<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 of AAVS1 gene AAVS1-sg1

<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> The target site of AAVS1 gene AAVS1-sg2

<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 (10)

1. an accurate NHEJ repair recombination screening gene, it is characterized in that: comprise the double sgRNA target site construction sequence that is used to be inserted in the screening/reporter gene sequence cut-off position, and the double sgRNA target site construction sequence includes being used for and being positioned at. The gene target sequences upstream and downstream of the blocking position constitute the three-base sequences of the two sgRNA sequences and the two PAM sequences respectively connected to the corresponding sgRNA sequences. 2 . An accurate NHEJ repair recombination screening gene according to claim 1 , wherein the double sgRNA target site construction sequence further comprises an unrelated sequence for connecting two PAM sequences. 3 . 3. An accurate NHEJ repair recombination screening gene according to claim 2, wherein the irrelevant sequence is 130-150bp. 4. An accurate NHEJ repair screening reporter vector, characterized in that: it comprises a screening reporter gene expression cassette and an sgRNA expression cassette, and the transcription region of the screening reporter gene expression cassette comprises an accurate NHEJ repair recombination screening gene sequence; the accurate NHEJ repair The recombinant screening gene sequence includes a double sgRNA target site construction sequence for inserting into the interrupted position of the drug screening gene sequence in the transcription region, and the dual sgRNA target site construction sequence includes a dual sgRNA target site construction sequence for being positioned upstream of the blocking position and The downstream drug screening gene target sequences consist of three nucleotide sequences of two sgRNA sequences and two PAM sequences respectively connected to the corresponding sgRNA sequences. The double sgRNA target sites respectively use the sgRNA transcribed by the sgRNA expression cassette as the target for the precise NHEJ repair. Guide sequences for recombinant screening gene sequences. 5. A kind of accurate NHEJ repair screening report vector according to claim 4, is characterized in that: described double sgRNA target site construction sequence also comprises the irrelevant sequence for connecting two PAM sequences; Described accurate NHEJ repair screening report The vector performs precise NHEJ repair on the CRISPR/Cas9-mediated double sgRNA target site break, restores the drug screening gene sequence in the transcription region of the screening reporter gene expression cassette, and expresses the corresponding drug resistance. 6. A kind of accurate NHEJ repair screening report carrier according to claim 4, is characterized in that: the transcription region of described screening reporter gene expression cassette also comprises fluorescent reporter gene sequence, and the fluorescent reporter gene sequence and described drug screening gene sequence are co-expressed, and the fluorescent reporter gene sequence is located downstream of the drug screening gene sequence. 7. An accurate NHEJ repair screening reporter vector according to claim 4 or 6, characterized in that: the screening reporter gene expression cassette further comprises a promoter and a termination sequence located on both sides of the transcription region. 8. A system for screening and enriching long-fragment precise deletion cells, characterized in that it comprises an accurate NHEJ repair screening reporter vector and a target editing cell co-transfected with the screening reporter vector for accurately deleting a selected site in the genome of the cell. The long fragment targeting vector between the two; the precise NHEJ repair screening reporter vector includes a screening reporter gene expression cassette and an sgRNA expression cassette, and the transcription region of the screening reporter gene expression cassette includes an accurate NHEJ repair recombination screening gene sequence; The precise NHEJ Repairing the recombination screening gene sequence includes a double sgRNA target site construction sequence for inserting into the interrupted position of the drug screening gene sequence in the transcription region, and the dual sgRNA target site construction sequence includes a double sgRNA target site construction sequence for being positioned upstream of the blocking position. and the downstream drug screening gene target sequences constitute two sgRNA sequences three-base sequences and two PAM sequences respectively connected to the corresponding sgRNA sequences, and the double sgRNA target sites respectively use the sgRNA transcribed by the sgRNA expression cassette as the target for the precise NHEJ. Repair the guide sequence of the recombinant screening gene sequence; the length of the long fragment is greater than or equal to the double sgRNA target site construction sequence. 9 . The system for screening and enriching long fragments for precise deletion of cells according to claim 8 , wherein the long fragments are 100-1300 bp. 10 . 10 . The application of an accurate NHEJ repair screening reporter vector as claimed in claim 4 in enriching gene knockout cells with precise deletion of biallelic long fragments. 11 .

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