Universal gene editing positive cell screening reporter vector system mediated by crisper / cas9

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a CRISPR / Cas9-mediated universal gene editing positive cell screening report system. The system comprises an animal cell universal screening report vector and a CRISPR / Cas9 expression vector; a genome is targeted by the CRISPR / Cas9 expression vector, and a universal sgRNA guided Cas9 protein targets double fluorescent genes and a resistance gene screening report expression frame on the vector skeleton, and after homologous recombination repair, the fluorescent gene and the drug resistance gene expression frame are repaired. The universal genome editing screening report system can reflect the nuclease activity of the transfected cells in real time, and can be widely used for simple and efficient screening and enrichment of positive cells of genome editing at any site of cells.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a CRISPR / Cas9-mediated universal gene editing positive cell screening reporter system. Background Technology

[0002] CRISPR / Cas9 nuclease technology, as the third-generation gene editing technology following zinc finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), originates from the type II system of *Streptococcus pyogenes*. The CRISPR / Cas9 system mainly consists of two parts: clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (Cas9). The Cas9 protein, relying on guide RNA (gRNA), recognizes and cleaves the target DNA sequence, subsequently generating double-strand breaks (DSBs), thereby modifying the specific DNA sequence. Repair of DSBs in cells mainly involves non-homologous end joining (NHEJ) and homology-directed repair (HDR). Among these, the NHEJ repair mechanism is the most important in mammals. Its key feature is its ability to repair DNA molecule breaks through non-specific end-joining in the absence of donor DNA or repeating sequences at both ends of DSBs. However, NHEJ repair tends to introduce small amounts of nucleotide insertions or deletions (indels) at DSBs, making it a non-precise repair pathway. Homologous recombination repair, in the presence of donor DNA, relies on donor DNA as a template for precise repair. However, HDR repair efficiency is far lower than NHEJ repair, significantly limiting its widespread application in precise genome engineering. Therefore, improving HDR efficiency is of great value in genome editing research. Currently, many effective strategies have been developed to enhance HDR repair efficiency. These strategies include: inhibiting the NHEJ pathway, such as using Scr7 inhibitors, knocking out Ku70 / 80 or Ligase IV; promoting the HDR pathway, such as overexpressing Rad51 / Rad52, using agonists Brefeldin A and L755507; and optimizing the repair template, such as increasing the length of the template homologous arm and using single-stranded DNA (ssDNA) donor templates. In addition, selecting an appropriate cell cycle (such as prolonging the S / G2 phase) has also been shown to increase HDR efficiency.

[0003] Screening and enriching gene-edited positive cells is a complex and time-consuming process. Conventional screening and enrichment methods use plasmids with selection tag genes (e.g., fluorescently labeled genes or drug selection genes), but this method can only screen transfected positive cells and cannot determine whether the intracellular genome has been modified. Subsequently developed reporter vectors based on NHEJ and HDR repair mechanisms utilize the characteristics of nuclease-positive cells to improve the enrichment efficiency of gene-edited positive cells. Among them, NHEJ-based reporter vectors have lower repair efficiency (only up to two-thirds of the repair products can restore the reporter gene function), making them more suitable for enriching cells with insertions and deletions. While HDR-based reporter vectors can be used to enrich gene-edited positive cells, their limited screening method and complex vector structure restrict their widespread application. Summary of the Invention

[0004] The purpose of this invention is to provide a CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system. This screening reporter system screens nuclease-positive cells by fluorescence sorting or drug screening and can be applied to the screening and enrichment of gene-editing positive cells at any gene-editing site in animal cells, so as to achieve simpler and more efficient enrichment to obtain gene-editing positive cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system, comprising a universal animal cell screening reporter vector and a CRISPR / Cas9 expression vector;

[0007] The universal animal cell screening reporter vector includes a universal sgRNA expression cassette, a dual-fluorescent gene and resistance gene screening reporter expression cassette, and a resistance gene homologous recombination repair template.

[0008] The transcriptional region of the universal sgRNA expression cassette includes a guide RNA that targets the selection tag coding sequence of the vector itself;

[0009] The transcriptional region of the dual-fluorescent gene and resistance gene screening reporter expression cassette includes: an upstream fragment homologous to the resistance gene coding region, a downstream fragment homologous to the resistance gene coding region, and the gene coding sequence of a first fluorescent protein (which, by expressing the corresponding fluorescent protein, indicates the gene coding sequence of the plasmid transfected into the cell, thereby reporting transfected positive cells), a cleavage peptide coding sequence, and the gene coding sequence of a second fluorescent protein (which, by expressing the corresponding fluorescent protein, indicates that the plasmid transfected into the cell has been edited by the Cas9 protein with nuclease cleavage activity and successfully repaired, thereby reporting nuclease positive cells) located between the upstream and downstream fragments. The upstream and downstream fragments are used for homologous recombination repair sequences.

[0010] The CRISPR / Cas9 expression vector includes an sgRNA expression cassette for expressing the target sequence of the guide cell genome and an expression cassette for expressing the Cas9 protein (hereinafter referred to as the Cas9 expression cassette), wherein the guide RNA recognizes the genome derived from the gene-editing target cell.

[0011] Preferably, the target sequence recognized by the sgRNA transcribed by the universal sgRNA expression cassette is a homologous sequence to the gene coding sequence of the first fluorescent protein.

[0012] Preferably, the upstream fragment homologous to the resistance gene coding region, the gene coding sequence of the first fluorescent protein, the downstream fragment homologous to the resistance gene coding region, the cleavage peptide coding sequence, and the gene coding sequence of the second fluorescent protein share the same open reading frame. The dual fluorescent gene and the resistance gene screening reporter expression cassette share the same open reading frame, which is separated by a stop codon set between the gene coding sequence of the first fluorescent protein and the downstream fragment.

[0013] Preferably, the upstream fragment corresponds to a 290bp fragment of the positive sequence of the homologous recombination repair template sequence of the resistance gene, and the downstream fragment corresponds to a 290bp fragment of the negative sequence of the homologous recombination repair template sequence of the resistance gene; the sum of the sizes of the upstream and downstream fragments is less than the size of the coding region of the resistance gene (600bp).

[0014] Preferably, the first fluorescent protein is a red fluorescent protein (e.g., mCherry protein), and the second fluorescent protein is a green fluorescent protein (e.g., EGFP protein). The system expresses only red fluorescent protein before repair and only green fluorescent protein after repair.

[0015] Preferably, the upstream and downstream fragments, after homologous recombination repair, are used to express the sequence for puromycin resistance.

[0016] Preferably, the gene coding sequence of the second fluorescent protein is located downstream of the downstream fragment, and the transcriptional region of the dual fluorescent gene and resistance gene screening reporter expression cassette further includes a spliced ​​peptide sequence for connecting the gene coding sequences of the downstream fragment and the second fluorescent protein.

[0017] Preferably, the homologous recombination repair template for the resistance gene is selected from the sequence corresponding to the coding region of the resistance gene that does not contain a start codon.

[0018] The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system was prepared by the following steps:

[0019] 1) Using a vector carrying the resistance gene (such as pSpCas9n(BB)-2A-Puro, PX462, addgene#48141) as a template, the resistance gene (PuroR) sequence (iPuroR) without the start codon (ATG) was amplified by PCR. The iPuroR sequence was then integrated with the backbone vector pcDNA3.1 through enzyme digestion and ligation to construct the iPuro-pcDNA3.1 vector.

[0020] 2) The sequence of the universal guide RNA (sgME) is ligated to a backbone vector (e.g., pX330-U6-Chimeric_dBbsI-CBh-hSpCas9) to obtain a universal sgRNA expression cassette; then, it is ligated to the expression vector (iPuro-pcDNA3.1 vector) and cloned to obtain the expression vector iPuro-U6-sgME-pcDNA3.1, which includes a homologous recombination repair template for the resistance gene and a universal sgRNA (sgME) expression cassette.

[0021] 3) Clones containing the upstream fragment homologous to the coding sequence of the resistance gene (Puro.L) 1-290 ), and downstream fragments homologous to the resistance gene coding sequence (Puro.R 308-597 The recombinant fragments were then ligated into the expression vector (iPuro-pcDNA3.1 vector) and integrated with the cloned gene coding sequence of the first fluorescent protein. The gene coding sequence of the first fluorescent protein was then inserted between the upstream and downstream fragments to obtain the dual-fluorescent gene and resistance gene selection reporter gene recombinant expression vector (iPuro-U6-sgME-pcDNA3.1-Puro-L). 1-290 -Puro-R 308-597 -T2A-EGFP vector);

[0022] 4) Insert the cloned gene coding sequence containing the first fluorescent protein (e.g., the mCherry sequence) into the sequence between the upstream and downstream fragments above to obtain a universal gene-edited positive cell screening reporter vector (iPuro-U6-sgME-pcDNA3.1-Puro-L). 1-290 -mCherry-Puro-R 308-597 -T2A-EGFP vector), abbreviated as UPME (Universal Puromycin and mCherry or EGFP vector);

[0023] Preferably, the above preparation method further includes the following steps: designing fitting primers according to the target gene target site sequence, annealing the fitting primers to obtain primer annealed double strands, connecting the primer annealed double strands to the vector backbone containing the above nuclease expression cassette, and using the primer annealed double strands to form the above sgRNA expression cassette in the vector backbone to obtain a site-specific CRISPR / Cas9 expression vector.

[0024] Based on the same inventive concept, this invention also provides the application of the CRISPR / Cas9-mediated universal gene editing positive cell screening reporter system in screening gene editing positive cells, wherein the cells are animal cells.

[0025] Based on the same inventive concept, this invention also provides a method for screening CRISPR / Cas9-mediated gene editing positive cells, comprising the following steps:

[0026] 1) Transfect the aforementioned CRISPR / Cas9-mediated universal gene editing positive cell screening reporter system into gene editing target cells;

[0027] 2) Flow cytometry is used to sort nuclease-positive cells expressing green fluorescence 48–72 hours after transfection, and then the cells are enriched and cultured. Alternatively, puromycin is used to screen for nuclease-positive cells 48–72 hours after transfection, and then the cells are enriched and cultured.

[0028] Preferably, the final concentration of the drug (e.g., puromycin) added to the transfected cell culture system is 0.2–5 μg / mL (depending on the actual resistance of different cells to the drug).

[0029] Preferably, the CRISPR / Cas9 expression vector and the selection reporter vector are co-transfected into mammalian cells for expression. The dual fluorescent gene and the resistance gene selection reporter expression cassette of the universal selection reporter vector are cleaved by the Cas9 protein expressed by the CRISPR / Cas9 expression vector (the cleavage site is located in the first fluorescent coding sequence, and the Cas9 protein targets specific target sites on the animal cell genome) and homologous recombination repair is completed. At the same time, the expression of the resistance gene and the second fluorescent reporter gene is restored while the expression of the first fluorescent protein reporter gene is turned off (deleted from the transcriptional region of the dual fluorescent gene and resistance gene expression cassette due to cleavage and homologous recombination repair).

[0030] Preferably, after animal cells are transfected with the vector of the universal gene-editing positive cell screening reporter system, nuclease-positive cells exhibit green fluorescent markers (e.g., EGFP) and drug resistance (e.g., puromycin); while nuclease-negative cells (i.e., transfected positive cells) exhibit red fluorescent markers (e.g., mCherry) and do not exhibit drug resistance. Screening of nuclease-positive cells is achieved through the expression of different fluorescent reporter genes. Simultaneously, the ratio of cells with red fluorescent markers (e.g., mCherry+) to the total number of cells characterizes the efficiency of cell transfection, and the ratio of cells with green fluorescent markers (e.g., EGFP+) to the total number of cells characterizes the efficiency of nuclease cleavage activity (i.e., the screening reporter vector repair efficiency).

[0031] The present invention has the following beneficial effects:

[0032] The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system (UPME) developed in this invention can induce resistance in transfected cells through homologous recombination repair of antagonistic gene sequences (e.g., Puromycin), causing the cells to lose the expression of the first fluorescence (e.g., mCherry) while simultaneously beginning to express the expression of the second fluorescence (e.g., EGFP). This system allows for rapid and convenient detection of nuclease activity in target cells via fluorescence sorting or drug screening, and the selection of nuclease-positive cells to improve the enrichment efficiency of gene-editing positive cells. The screening reporter system employs a homologous recombination HDR repair method, which can be applied to the enrichment of gene knock-in positive cells as well as gene knockout positive cells. Furthermore, the screening reporter system can be widely applied to various cell types in mammals. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the UPME system in operation.

[0034] Figure 2The image shows the agarose gel electrophoresis identification of the enzyme digestion products of the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector. Lane M: Trans2K plus II DNA Marker; Lanes 1 and 2: Vector digested with Bsa I.

[0035] Figure 3 The image shows the Sanger sequencing results for the pX330-sgME-CBh-hSpCas9 vector.

[0036] Figure 4 This is an agarose gel electrophoresis image of the U6-sgME-scaffold fragment. Lane M: Trans2K DNA Marker; Lanes 1 and 2: Amplification results of the U6-sgME-scaffold sequence.

[0037] Figure 5 For Puro.L 1-290 andPuro.R 310-600 Fragment agarose gel electrophoresis image. In Figure A, lane M: Trans2K DNA Marker; lanes 1 and 2: Puro.L. 1-290 Sequence amplification results. In Figure B, lane M: Trans2K DNA Marker; lanes 1 and 2: Puro.R 308-597 The results of sequence amplification.

[0038] Figure 6 pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA vector (plasmid) restriction enzyme digestion identification diagram, where lane M: Trans2K plus II DNA Marker; lanes 1 and 2: plasmid digested with BamHI and NotI.

[0039] Figure 7 This is an agarose gel electrophoresis image of the iPuroR fragment. Lane M: Trans2K DNA Marker; Lanes 1 and 2: Amplification results of the U6-sgME-scaffold sequence.

[0040] Figure 8 pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA-iPruoR vector restriction enzyme digestion identification diagram, where lane M: Trans2K plus II DNA Marker; lanes 1 and 2: plasmids digested with Bgl II and HindIII.

[0041] Figure 9 pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA-iPruoR-U6-sgME vector restriction enzyme digestion identification diagram, where lane M: Trans2K plus II DNA Marker; lanes 1 and 2: plasmids digested with Bgl II and Hind III.

[0042] Figure 10 pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA-iPruoR-U6-sgME vector map.

[0043] Figure 11 This is a Sanger sequencing result of the pX330-U6-sgNUDT5-CBh-hSpCas9 vector.

[0044] Figure 12 The spectrum of the pX330-U6-sgNUDT5-CBh-hSpCas9 vector.

[0045] Figure 13 This is an agarose gel electrophoresis image of the NUDT5 gene donor template fragment. Lane M: Trans2K DNA Marker; Lanes 1 and 2: Amplification results of the NUDT5 gene donor template sequence.

[0046] Figure 14 This is a map of the pPuro-T2A-EGFP vector.

[0047] Figure 15 This document outlines the operational procedures for enriching gene-editing-positive cells using the UPME-based system.

[0048] Figure 16 The results show the cell morphology of the transfection-positive group (PTG-KO) and the screening reporter group (UPME-KO) in gene knockout cells, and the transfection-positive group (PTG-KI) and the screening reporter group (UPME-KI) in gene knockin cells, 60 hours after cell transfection.

[0049] Figure 17The results show the cell morphology of the transfection-positive group (PTG-KO) and the screening reporter group (UPME-KO) in gene knockout cells and the transfection-positive group (PTG-KI) and the screening reporter group (UPME-KI) in gene knockin cells 72 hours after screening with puromycin.

[0050] Figure 18 This figure shows the deep sequencing results of gene editing efficiency in NUDT5 knockout cells obtained using FACS sorting and drug screening, specifically the transfection-positive group (PTG-KO) and the screening reporter system group (UPME-KO).

[0051] Figure 19 This figure shows the deep sequencing results of gene editing efficiency in the PTG-KI and UPME-KI groups, obtained by FACS sorting and drug screening and knocking in NUDT5 sites into cells. Detailed Implementation

[0052] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0053] Example 1: Construction of a CRISPR / Cas9-mediated universal gene editing positive cell screening reporter system (UPME).

[0054] 1. Cloning a universal sgRNA (U6-sgME-scaffold) expression cassette

[0055] 1.1 Target Site Selection

[0056] Based on the principles of UPME vector pre-design, sgRNAs were designed using the second fluorescent protein gene coding sequence (mCherry) as a template and online design software (http: / / crispr.mit.edu / ) (design principles: GC content 40%-60%; cleavage efficiency greater than 65%; no target in the genomes of mammals such as humans, mice, pigs, cattle, and sheep; very low off-target effects). The selected target sequence (SEQ ID NO.1) is: 5'-GCGTTCGTACTGTTCCACGA TGG -3' (TGG is the PAM recognition site). That is, the target sequence contained in the universal sgRNA (sgME) expressed in the UPME vector is: 5'-GCGTTCGTACTGTTCCACGA-3'.

[0057] 1.2 Construction of pX330-sgME-CBh-hSpCas9 vector

[0058] Fitting primers were designed targeting the target site, using pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene#42230) as the backbone vector. The designed fitting primer sequences are as follows.

[0059] sgME-F (Bbs I) (SEQ ID NO.2): 5'-caccGCGTTCGTACTGTTCCACGA-3';

[0060] sgME-R (Bbs I) (SEQ ID NO. 3): 5'-aaacTCGTGGAACAGTACGAACGC-3'.

[0061] Use 50 pmol each of primers sgME-F (Bbs I) and sgME-R (Bbs I), mix well, and place in PCR for annealing fitting annealing (see Table 1). The product is a primer-annealed double strand with complementary pairing to the sticky ends after Bsa I cleavage.

[0062] Table 1 Primer annealing procedure

[0063]

[0064] The pX330-U6-Chimeric_BB-CBh-hSpCas9 vector was digested with BBSI (see Table 2 for the digestion system) in a 37°C water bath for 0.5–1 h. The digested fragments were detected by agarose gel electrophoresis, and the target band corresponding to the vector backbone was recovered using a DNA recovery kit (see Table 2 for details). Figure 2 ).

[0065] Table 2 Single enzyme digestion system

[0066]

[0067] The recovered vector backbone product and the primer-annealed product were ligated using T4 DNA ligase (ligation system shown in Table 3). The ligation reaction conditions were 25℃ for 15-30 min. Then, the mixture was transformed into *E. coli* DH5α competent cells and ligated using LB / AMP. + Cultured in screening medium.

[0068] Table 3 Connection System

[0069]

[0070] After expanding the culture of positive clones, plasmids were extracted and sent to Qingke Biotechnology Co., Ltd. for Sanger sequencing identification. Correctly sequenced plasmids (see...) Figure 3 ).

[0071] 1.3 Amplification of the U6-sgME-scaffold sequence

[0072] Using pX330-sgME-CBh-hSpCas9 plasmid as a template, primers were designed for PCR cloning and amplification of the U6-sgME-scaffold sequence (PCR amplification system is shown in Table 4). The primer sequences are as follows:

[0073] U6-F(Mlu I)(SEQ ID NO.4):

[0074] U6-R(Spe I)(SEQ ID NO.5):

[0075] Table 4. U6-sgME-scaffold sequence cloning and amplification system

[0076]

[0077] PCR products were detected by agarose gel electrophoresis, and the target band was recovered using a DNA recovery kit (see [link to kit]). Figure 4 This involves amplifying the U6-sgME-scaffold sequence and adding Mlu I and Spe I restriction endonuclease recognition sites to both ends of the sequence.

[0078] 2. Construction of pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA vector

[0079] 2.1 Amplification of Puro.L 1-290 andPuro.R 308-597 sequence

[0080] Primers were designed and synthesized using the pSpCas9n(BB)-2A-Puro vector (Addgene#48141) as a template for PCR amplification (see Table 5). The primer sequences are as follows:

[0081] Puro.LF(Hind III)(SEQ ID NO.6):

[0082] Puro.LR(BamH I)(SEQ ID NO.7):

[0083] Puro.RF(Not I)(SEQ ID NO.8): 5'-atGCGGCCGCGGCCGCGCAGCAA-3';

[0084] Puro.RR(Xho I)(SEQ ID NO.9):

[0085] In the primer sequences above, lowercase letters represent protective bases, and uppercase bold italic letters represent restriction endonuclease recognition sites. The designed primers were sent to Qingke Biotechnology Co., Ltd. for synthesis (the same applies below).

[0086] Table 5 PCR cloning amplification system

[0087]

[0088] PCR products were detected by agarose gel electrophoresis, and the target band was recovered using a DNA recovery kit (see [link to kit]). Figure 5 ).

[0089] 2.2 Construction of pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA vector

[0090] Using the pSSA-PMG vector (Addgene#208857) as the backbone vector, Puro.L was integrated by double digestion with Hind III and BamHI. 1-290 The sequence was then ligated and integrated into Puro.R using Not I and Xho I double digestion. 308-597 The double enzyme digestion system is shown in Table 6, and the ligation system is shown in Table 3. The ligation product was transformed into DH5α competent cells, and positive clones were screened using LB / AMP+ medium. After plasmid extraction, double enzyme digestion was used for identification (see Table 3). Figure 6 The correctly sequenced plasmid is pcDNA3.1-CMV-Puro.L. 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA vector.

[0091] Table 6 Double enzyme digestion system

[0092]

[0093] 3.pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 Construction of the -T2A-EGFP-polyA-iPruoR vector

[0094] 3.1 Amplification of the iPuroR sequence

[0095] Primers were designed and synthesized using the pSpCas9(BB)-2A-Puro vector plasmid (Addgene 48139) as a template for PCR cloning and amplification. The primer sequences are as follows:

[0096] iPuroR-F(Bgl I)(SEQ ID NO.10):

[0097] iPuroR-R(Mlu I)(SEQ ID NO.11):

[0098] After PCR cloning and amplification, the samples were detected by agarose gel electrophoresis, and the target bands were recovered using a DNA recovery kit (see [link to kit]). Figure 7 This process completes the cloning of the homologous recombination repair template for the resistance gene (the ATG coding sequence of the puromycin resistance gene deletion promoter), with restriction endonuclease Bgl I and Mlu I recognition sites added to both ends, resulting in the iPuroR sequence.

[0099] 3.2 Construction of pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 Construction of the -T2A-EGFP-polyA-iPruoR vector

[0100] pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 The T2A-EGFP-polyA vector and iPuroR sequence gel-recovered products were double-digested with Bgl II and Mlu I restriction endonucleases, respectively (see Table 6 for the double digestion system), and then ligated using T4 DNA ligase.

[0101] The ligation product was transformed into DH5α competent cells, and positive clones were screened using LB / AMP+ medium. After plasmid extraction, double enzyme digestion was used for identification (see [link to relevant documentation]). Figure 8 The correct plasmid is pcDNA3.1-CMV-Puro.L 1-290-mCherry-Puro.R 308-597 -T2A-EGFP-polyA-iPruoR vector.

[0102] 4. Construct pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA-iPuroR-U6-sgME vector (UPME vector)

[0103] With pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 The -T2A-EGFP-polyA-iPuroR vector was used as the backbone vector, and Mlu I and Spe I restriction endonucleases were used to target pcDNA3.1-CMV-Puro.L. 1-290 -mCherry-Puro.R 308-597 The T2A-EGFP-polyA-iPuroR vector and the U6-sgME-scaffold amplification product were double-digested with enzymes (see Table 6), and then ligated using T4 DNA ligase.

[0104] The ligation product was transformed into DH5α competent cells, and positive clones were screened using LB / AMP+ medium. After plasmid extraction, double enzyme digestion was used for identification (see [link to relevant documentation]). Figure 9 The correct plasmid is pcDNA3.1-CMV-Puro.L 1-290 -mCherry-Puro.R 308-597 -T2A-EGFP-polyA-iPruoR-U6-sgME vector (see also) Figure 10 ).

[0105] 5. Constructing site-specific CRISPR / Cas9 targeting vectors

[0106] Taking the NUDT5 gene as an example, based on its genome sequence (NCBI NM_004097.3), primers were designed using the CRISPR Dsign website (http: / / crispr.mit.edu / ).

[0107] The pX330-U6-Chimeric_BB-CBh-hSpCas9 vector was digested with Bbs I (see Table 6 for the digestion system), and the digestion product was recovered and then ligated with the matching primers for the NUDT5 gene.

[0108] The fitted primer sequences are as follows:

[0109] NUDT5-F (Bbs I) (SEQ ID NO. 12): 5'-caccGTGAAGTGTTCTCTGCAGCA-3';

[0110] NUDT5-R (Bbs I) (SEQ ID NO. 13): 5'-aaacTGCTGCAGAGAACACTTCAC-3'.

[0111] The ligation product was transformed into DH5α competent cells, and positive clones were screened using LB / AMP+ medium. Plasmids were extracted and sequenced using Sanger sequencing (see [link]). Figure 11 The correctly sequenced plasmid is the pX330-U6-sgNUDT5-CBh-hSpCas9 vector (see [link]). Figure 12 ).

[0112] 6. Donor templates for amplifying specific sites

[0113] Taking the NUDT5 gene as an example, based on the designed target site sequence, 800-1000 bp homologous arm sequences were amplified by PCR on both sides of the target site. The PCR amplification program is shown in Table 5. Simultaneously, specific primers were designed to replace the PAM sequence on the target sequence recognized by sgRNA on the NUDT5 genome with the EcoRI restriction endonuclease recognition site. The two fragments were fused into a single DNA sequence using overlap extension PCR to obtain a 2140 bp donor template (see Table 5). Figure 13 ).

[0114] The PCR amplification primer sequences are as follows:

[0115] NUDT5-F (SEQ ID NO.14): 5'-acAGATCTCAAACCGTCTTCTCCATTC-3';

[0116] NUDT5-R (SEQ ID NO. 15): 5'-taGGATCCCTTCTGGCTTCTGTTATC-3';

[0117] NUDT5(EcoR I)-F (SEQ ID NO. 16): 5'-GTGTTCTCTGCAGCAGAA TTCTGACCGCGACACCTGTCAC-3';

[0118] NUDT5(EcoR I)-R (SEQ ID NO. 17): 5'-GTCGCGGTCAGAATTCTG CTGCAGAGAACA CTTCACTATG-3'.

[0119] 7. Identification of positive screening reporter vector (pPuro-T2A-EGFP)

[0120] Transfection with the positive selection reporter vector pPuro-T2A-EGFP (Lyu M, Sun Y, Yan N, et al. Efficient CRISPR / Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters[J]. International Journal of Biological Macromolecules: Structure, Function and Interactions, 2023:243.) (See also...) Figure 14 After transfection of cells with this vector, they can express the puromycin resistance gene (PuroR) and the green fluorescent protein gene (EGFP) normally. Therefore, transfection with this vector and a target vector donor with a specific site can be used as a transfection positive group.

[0121] Example 2: Working principle and process of the UPME system

[0122] 1. Working principle

[0123] The UPME screening reporter system constructed in Example 1 is a CRISPR / Cas9-mediated gene editing positive cell screening reporter vector system. The UPME screening reporter system mainly consists of two parts: a universal UPME screening reporter vector and a site-specific CRISPR / Cas9 expression vector (e.g., pX330-U6-sgNUDT5-CBh-hSpCas9 vector).

[0124] A universal UPME screening reporter vector mainly includes a universal sgRNA expression cassette (e.g., sgRNA designed based on the mCherry encoding gene), a homologous recombination repair template for resistance genes (e.g., a homologous recombination template designed based on the PuroR encoding gene), a dual fluorescent reporter system (e.g., a red / green fluorescent reporter system based on mCherry / EGFP), and a drug resistance (e.g., a puro-based puromycin resistance) expression cassette.

[0125] After cell transfection using the UPME reporter system, the UPME reporter vector expresses a sgRNA complex (sgME) targeting the vector itself. Site-specific CRISPR / Cas9 expression vectors express sgRNA and Cas9 protein targeting specific genome-specific sequences. Subsequently, the sgRNA transcribed from the UPME reporter vector guides the Cas9 protein to recognize and cleave the UPME reporter vector's own DNA sequence, while the sgRNA targeting specific sequences guides the Cas9 protein to recognize and cleave genome-specific target sequences.

[0126] See Figure 1 After the universal UPME screening reporter vector is transfected into cells, along with its dual-fluorescent gene and resistance gene screening reporter expression cassette located in Puro.L 1-290 andPuro.R 308-597 While the target sequence region on the coding sequence of the first fluorescent protein gene is cleaved, HDR repair can be performed using the homologous recombination repair template sequence of the resistance gene on the vector itself. As a result, while the red fluorescent protein mCherry sequence is deleted, the coding sequences of the puromycin resistance gene and the green fluorescent protein EGFP gene are translated normally, thus achieving the expression of puromycin resistance and green fluorescence under CMV initiation. If the above cleavage does not occur, the UPME screening reporter vector can only normally express Puro.L. 1 -290 The nucleotide sequence preceding the red fluorescent protein mCherry allows for normal expression of red fluorescence (representing positive transfection) under CMV initiation. Therefore, by co-transfecting a CRISPR / Cas9 expression vector with a specific site, the universal UPME screening reporter vector itself can achieve puromycin resistance repair expression and dual-fluorescence reporter system operation (two fluorescence conversions) through HDR homologous recombination repair. This allows it to be used for flow cytometry sorting or puromycin drug screening to enrich gene-edited positive cells.

[0127] 2. Specific enrichment experiment process (see...) Figure 15 )

[0128] (1) Design CRISPR / Cas9 targeting vectors and corresponding donor vectors for specific gene editing site sequences in the cell genome;

[0129] (2) Co-transfect cells (e.g., HEK293T cells) with the constructed CRISPR / Cas9 targeting vector, donor vector and UPME screening reporter vector;

[0130] (3) 48-72 hours after transfection, the proportion of cells exhibiting nuclease cleavage activity was detected by flow cytometry (the ratio of nuclease-positive cells to transfected positive cells after transfection with the screening reporter vector = screening reporter vector repair efficiency / plasmid vector cell transfection efficiency), or nuclease-positive cells were obtained by flow cytometry sorting.

[0131] (4) Alternatively, 48-72 hours after transfection, puromycin can be added to the cell culture medium. After 3-5 days of puromycin screening, the drug is withdrawn and replaced with normal cell culture medium to obtain the screened cells (referring to nuclease-positive cells).

[0132] (5) Dilute the selected cells using the limiting dilution method and then plate them into 96-well plates.

[0133] (6) After 7-10 days, cell clones were randomly screened and positive cell clones with gene editing at the target editing site of the genome were identified by PCR.

[0134] (7) Transfer positive monoclonal cells to 24-well plates for expanded cell culture. For example, for HEK293T cells, use DMEM medium containing 10% fetal bovine serum and 100 μg / mL penicillin / streptomycin as the cell culture medium and culture them in a cell culture incubator at 37°C and 5% CO2.

[0135] Example 3: Specific application of the UPME system in enriched CRISPR / Cas9-mediated gene editing positive cells

[0136] Taking the human NUDT5 gene locus as an example, the UPME system was used to enrich cells with gene editing (knockout or knock-in) at this locus.

[0137] A CRISPR / Cas9 expression vector (pX330-U6-sgNUDT5-CBh-hSpCas9) targeting the NUDT5 site was constructed, along with a UPME screening reporter vector and a gel recovery product of the homologous repair template (enriched gene knock-in positive cells). The construction and identification methods are described in Example 1.

[0138] For cells enriched to be gene knockout positive, once the cells reached a suitable density, HEK293T cells were co-transfected with the positive selection reporter vector pPuro-T2A-EGFP and pX330-U6-sgNUDT5-CBh-hSpCas9 as the gene knockout control group (PTG-KO). HEK293T cells were co-transfected with the UPME vector and pX330-U6-sgNUDT5-CBh-hSpCas9 as the gene knockout experimental group (UPME-KO).

[0139] For cells enriched for gene knock-in positive, once the cells reached a suitable density, HEK293T cells were co-transfected with the positive selection reporter vector pPuro-T2A-EGFP, pX330-U6-sgNUDT5-CBh-hSpCas9, and a homologous repair template as the gene knock-in control group (PTG-KI). HEK293T cells were co-transfected with the UPME vector, pX330-U6-sgNUDT5-CBh-hSpCas9, and a homologous repair template as the gene knock-in experimental group (UPME-KI).

[0140] 60 hours after cell transfection (see Figure 16 For flow cytometry sorting, cells from each group were digested into 1.5 mL EP tubes and centrifuged at 1200 rpm for 5 minutes. The cell pellet was resuspended in PBS, and the labeled centrifuge tubes were placed in an ice box. FACS: Cells were sorted in the flow cytometer using the green fluorescence (EGFP+) channel, and cells expressing green fluorescence (EGFP+) were collected. Genomic DNA was extracted from each group of collected cells for genome editing efficiency assay.

[0141] For drug resistance screening, puromycin (3 μg / mL) was added to the cell culture medium of both the experimental and control groups for drug screening. After 72 hours of drug screening (see [link to study]), the results were obtained. Figure 17 After removing the drug, the enriched positive cells were observed using a fluorescence inverted microscope. Genomic DNA was then extracted from both groups of cells for genome editing efficiency testing.

[0142] For cells enriched by two different methods, flow cytometry fluorescence sorting and drug screening, genomic DNA was extracted as a template. Detection primers with different sequence barcodes (see Table 8) were used to amplify the region near the target site (162 bp). After gel recovery, the samples were sent to Xi'an Qingke Biotechnology Co., Ltd. for amplicon deep sequencing detection.

[0143] Table 7 Detection Primers

[0144]

[0145] After analyzing the amplicon deep sequencing results using the online software CRISPResso (http: / / www.crispresso.rocks), the efficiency of enrichment of the UPME system group and the transfection positive group into gene-edited positive cells was statistically analyzed; the results were statistically analyzed using GraphPadPrism 8.0.

[0146] For the enrichment of gene knockout positive cells, the analysis of positive cells obtained by FACS screening showed that the gene knockout positive cell efficiency obtained using the UPME system was 68.56%, while the efficiency of gene knockout positive cells obtained using the transfection positive group was 33.33%. The analysis of positive cells obtained by puromycin screening showed that the gene knockout positive cell efficiency obtained using the UPME system was 91.38%, while the efficiency of gene knockout positive cells obtained using the transfection positive group was 63.35% (see [link to puromycin]). Figure 18 );

[0147] For the enrichment of gene knock-in positive cells, the analysis of positive cells obtained by puromycin screening showed that the gene knock-in positive cell efficiency obtained using the UPME system was 7.35%, while the gene knock-in positive cell efficiency obtained using the transfection positive group was 1.93%; the analysis of positive cells obtained by puromycin screening showed that the gene knock-in positive cell efficiency obtained using the UPME system was 4.26%, while the gene knock-in positive cell efficiency obtained using the transfection positive group was 14.11% (see...). Figure 19 The above results indicate that the UPME screening report system can efficiently enrich gene-editing positive cells.

[0148] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0149] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system, characterized in that, The system includes a universal screening reporter vector for animal cells and a CRISPR / Cas9 expression vector; The universal animal cell screening reporter vector includes a universal sgRNA expression cassette, a dual-fluorescent gene and resistance gene screening reporter expression cassette, and a resistance gene homologous recombination repair template. The transcriptional region of the universal sgRNA expression cassette includes a guide RNA that targets the selection tag coding sequence of the vector itself; The transcriptional region of the dual-fluorescent gene and resistance gene screening reporter expression cassette includes: an upstream fragment homologous to the resistance gene coding region, a downstream fragment homologous to the resistance gene coding region, and the gene coding sequence of the first fluorescent protein, the cleavage peptide coding sequence, and the gene coding sequence of the second fluorescent protein located between the upstream and downstream fragments. The upstream fragment and the downstream fragment are used for homologous recombination repair sequences. The CRISPR / Cas9 expression vector includes an sgRNA expression cassette for expressing a target cellular genome sequence and an expression cassette for expressing the Cas9 protein.

2. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The target sequence recognized by the sgRNA transcribed by the universal sgRNA expression cassette is homologous to the gene coding sequence of the first fluorescent protein.

3. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The upstream fragment homologous to the resistance gene coding region, the gene coding sequence of the first fluorescent protein, the downstream fragment homologous to the resistance gene coding region, the cleavage peptide coding sequence, and the gene coding sequence of the second fluorescent protein share the same open reading frame. The dual fluorescent gene and the resistance gene screening reporter expression cassette share the same open reading frame, which is separated by a stop codon set between the gene coding sequence of the first fluorescent protein and the downstream fragment.

4. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The upstream fragment corresponds to a 290bp segment of the positive sequence of the homologous recombination repair template sequence of the resistance gene, and the downstream fragment corresponds to a 290bp segment of the negative sequence of the homologous recombination repair template sequence of the resistance gene; the sum of the sizes of the upstream and downstream fragments is less than the size of the coding region of the resistance gene.

5. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The first fluorescent protein is red fluorescent protein, and the second fluorescent protein is green fluorescent protein. Before the repair, the system only expresses red fluorescent protein, and after the repair, it only expresses green fluorescent protein.

6. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The upstream and downstream fragments, after homologous recombination repair, are used to express sequences for puromycin resistance.

7. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The gene coding sequence of the second fluorescent protein is located downstream of the downstream fragment, and the transcriptional region of the dual fluorescent gene and resistance gene screening reporter expression cassette also includes a spliced ​​peptide sequence for connecting the gene coding sequences of the downstream fragment and the second fluorescent protein.

8. The CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to claim 1, characterized in that, The homologous recombination repair template for the resistance gene is selected from the sequence corresponding to the coding region of the resistance gene that does not contain a start codon.

9. The application of the CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system according to any one of claims 1 to 8 in screening gene-editing positive cells, characterized in that, The cells in question are animal cells.

10. A method for screening CRISPR / Cas9-mediated gene-edited positive cells, characterized in that, Includes the following steps: 1) Transfecting the gene-editing target cells with a CRISPR / Cas9-mediated universal gene-editing positive cell screening reporter system as described in any one of claims 1 to 8; 2) Flow cytometry is used to sort nuclease-positive cells expressing green fluorescence 48–72 hours after transfection, and then the cells are enriched and cultured. Alternatively, puromycin is used to screen for nuclease-positive cells 48–72 hours after transfection, and then the cells are enriched and cultured.

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