CN109082443A - A method of preparing can the cell model that breaks up to mature hepatic lineage of real-time detection mescenchymal stem cell - Google Patents

Abstract

The present disclosure provides a method for preparing a cell model capable of real-time detection of differentiation of mesenchymal stem cells into mature hepatic-like cells, the method comprising the following steps: S1, knocking in sgRNA and Cas9 targeting genes related to hepatic-like cells Mixing proteins to form RNP complexes; S2, forming AAV virus particles to be transfected; S3, mixing the suspension of stem cells with the suspension of the RNP complexes and performing electroporation to obtain a culture after electroporation; S4, transferring to the Add the suspension of AAV virus particles to be transfected to the material after electroporation to perform transfection to obtain a transfected culture; S5, perform monoclonal culture after limiting the transfected culture , through PCR and sequencing and screen out the monoclonal cell line that has undergone gene-directed knock-in. Through the above-mentioned technical solution, the present invention can realize the real-time monitoring of the differentiation state of the continuously passaged cells to mature hepatic-like cells during the preparation process of the stem cell medicine.

Description

It is a kind of prepare can real-time detection mescenchymal stem cell break up to mature hepatic lineage it is thin The method of born of the same parents' model

Technical field

This disclosure relates to field of biotechnology, and in particular, to it is a kind of prepare can real-time detection mescenchymal stem cell at The method of the cell model of ripe hepatic lineage differentiation.

Background technique

Mescenchymal stem cell (MSCs) has a wide range of applications as the target cell that a kind of novel gene is treated, excellent Gesture is as follows: (1) it is from a wealth of sources, it can be obtained from self or allosome, belong to homograft, be not related to ethics problem；(2) it is easy to train It supports, there is proliferative capacity, inside and outside keeps multi-lineage potential；(3) there is site of tissue damage of going back to the nest, a large amount of cells of secretion The ability of the factor.But it is current the study found that the difference of the individual difference of donor, differentiation potential caused by the difference at age of drawing materials The different clinical application for constraining mescenchymal stem cell as cell transplantation drug.In as stem cell drugs preparation process, very In difficult Induction Process in vitro judgement be divided into the cells ratio of mature hepatic lineage, setting Quality Control node, realize real time monitoring, Batch wise differences between differentiation cell product due to caused by differentiation ratio difference, to limit it as cell transplantation drug Clinical application.How to pass in vitro and monitors the differentiation state of mescenchymal stem cell in amplification and atomization in time and also become dry Difficult point and bottleneck in cell industry Quality Control.

CRISPR gene editing technology forms DNA double by carrying out accurate targeting shearing in genome specific site Chain notch (Double-strand breaks, DSBs) links (Non-homologous end by intracellular nonhomologous end Joining, NHEJ) it realizes to the knockout of gene in situ, pass through homologous recombination (Homology directed repair, HDR) In the presence of external source gene template, the introducing of point mutation and inserting for reparation and large fragment gene (such as reporter gene) are realized Enter.As described in patent document CN105985985A, slow virus is constructed using CRISPR/Cas9 system, to umbilical cord source Immunizing antigen B2M, inflammatory factor TNF-α in MSCs are knocked out, so that realizing reduces allosome mescenchymal stem cell immunogen The purpose of property.But in mammalian cells, NHEJ revision points account for dominant pattern, realize gene knockout than realizing gene knock-in It is easy very much.And the segment of gene knock-in is bigger, it is lower to integrate probability, and technology is realized more difficult.It has had not yet to see The report of large fragment gene knock-in is carried out in MSCs.

Summary of the invention

The purpose of the disclosure is to realize the real-time detection broken up to mescenchymal stem cell to mature hepatic lineage, and providing can be real When the cell model that breaks up to mature hepatic lineage of detection mescenchymal stem cell to carry out drug screening.

To achieve the goals above, present disclose provides it is a kind of prepare can real-time detection mescenchymal stem cell to mature liver sample The method of the cell model of cell differentiation, this method comprises the following steps: S1, will knock in position for hepatic lineage related gene SgRNA the and Cas9 albumen of point is mixed to form RNP compound；The hepatic lineage related gene includes ALB and/or AFP；Institute That states hepatic lineage related gene knocks in the last sense codon and termination that site is the hepatic lineage related gene Between codon；S2, it will be packaged into inserted with template DNA homologous recombination vector in AAV virus, and form AAV virus to be transfected Particle；The template DNA includes homologous for the left homology arm sequence for knocking in site of the hepatic lineage related gene and the right side Arm sequence, inserted with self cleavage 2A peptide-coding sequence and fluorescence egg between the left homology arm sequence and the right homology arm sequence White reporter gene is connected in series so that orientation forms coding after knocking in by hepatic lineage related gene, 2A peptide and fluorescin Fusion protein fusion；S3, the suspension of mescenchymal stem cell is mixed and is carried out with the suspension of the RNP compound Electricity turns, and obtains the culture after electricity turns；S4, electricity turn after 1-30 minute in, to it is described electricity turn after material in addition to Culture of the suspension of the AAV virion of transfection to carry out transfection in 4-30 hours, after being transfected；S5, by the transfection After culture Method of Limited Dilution afterwards carry out monoclonal culture, and by PCR and sequencing filter out carried out gene orientation knock in Monoclonal cell strain.

Through the above technical solutions, the present invention, which establishes one kind, efficiently to knock in long segment report base in mescenchymal stem cell The method of cause, in the feelings for the gene expression for not influencing hepatic differentiation maturity symbol object ALB gene and liver prematurity reference substance AFP It is in situ to be inserted into reporter gene under condition, can be in stem cell drugs preparation process it is possible thereby to construct, real-time monitoring induction differentiation The cell model of differentiation and maturation situation that breaks up to mature hepatic lineage of mescenchymal stem cell, can it is easy, quickly, intuitive inspection It surveys mescenchymal stem cell and induces the differentiation and maturation situation broken up to mature hepatic lineage, be convenient for mescenchymal stem cell as drug The overall process of preparation carries out Quality Control, analysis and quality judging, pushes the standard for establishing its preparation of industrialization, its clinic is promoted to turn Change.

Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.

Specific embodiment

The specific embodiment of the disclosure is described in detail below.It should be understood that described herein specific Embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.

Present disclose provides a kind of prepare can the cell membrane that breaks up to mature hepatic lineage of real-time detection mescenchymal stem cell The method of type, this method comprises the following steps: S1, the sgRNA and Cas9 for knocking in site that will be directed to hepatic lineage related gene Albumen is mixed to form RNP compound；The hepatic lineage related gene includes ALB and/or AFP；The hepatic lineage is related Gene is knocked between the last sense codon and terminator codon that site is the hepatic lineage related gene；S2, It will be packaged into inserted with template DNA homologous recombination vector in AAV virus, and form AAV virion to be transfected；The template DNA The left homology arm sequence and right homology arm sequence for knocking in site including being directed to the hepatic lineage related gene, the left side are homologous Inserted with self cleavage 2A peptide-coding sequence and fluorescent protein report gene between arm sequence and the right homology arm sequence, so that The fusion that the fusion protein that coding is connected in series by hepatic lineage related gene, 2A peptide and fluorescin is formed after knocking in must be oriented Gene；S3, the suspension of mescenchymal stem cell is mixed to the suspension of the RNP compound and is carried out electricity turn, after obtaining electricity turn Culture；S4, in 1-30 minutes, AAV virion to be transfected is added in the material after turning to the electricity after electricity turns Culture of the suspension to carry out transfection in 4-30 hours, after being transfected；S5, by the culture Method of Limited Dilution after the transfection Monoclonal culture is carried out afterwards, and the monoclonal cell strain for having carried out gene orientation and having knocked in is filtered out by PCR and sequencing.

SgRNA and Cas9 albumen is transferred in cell in such a way that electricity turns by the present invention RNP compound, and after electricity turns The suitable time in selection AAV virus the homologous recombination vector inserted with fluorescent protein report gene is transfected into cell In, come sgRNA, Cas9 albumen and the carrier inserted with template DNA commonly through CRISPR gene editing so that big The foreign gene of segment is able to orientation and knocks in target position.

Wherein, the hepatic lineage related gene is defined with Gene Symbol in ncbi database, specific to believe As shown in table 1, corresponding representativeness sgRNA sequence and left and right homology arm sequence are also shown in Table 1 below breath.Preferably, described SgRNA is as shown in SEQ ID NO.1 or 4；Correspondingly, the left homology arm sequence is as shown in SEQ ID NO.2 or 5；Correspondingly, The right homology arm sequence is as shown in SEQ ID NO.3 or 6.

Table 1

Wherein, optionally, the sgRNA has chemical modification group；The chemical modification group is preferably methyl chemistry Modification group or phosphorothioate chemical modification group；For sgRNA the and Cas9 albumen for knocking in site of hepatic lineage related gene Dosage molar ratio be 1:1 to 1:5.Wherein it is possible to use online sgRNA design tool (http://crispr.mit.edu/) The sequence for knocking in site design sgRNA of foundation hepatic lineage related gene is simultaneously synthesized.Wherein, 5 ' ends of sgRNA sequence Methyl (- O-Me) chemical modification group or phosphorothioate (- phosphorothioate) can be added with respectively with 3 ' end ends Chemical modification group.

It wherein, optionally, will be for sgRNA the and Cas9 albumen mixing for knocking in site of hepatic lineage related gene Time is 5-20 minutes, and temperature is 10-40 DEG C.

Wherein, optionally, in step S3, when the suspension of the mescenchymal stem cell is mixed with the RNP compound, relatively In every 10⁶A mescenchymal stem cell, with the meter of sgRNA, the dosage of the RNP compound is 1-50 μm of ol；Described In the suspension of mesenchymal stem cells, cell concentration is (1-5) × 10⁷A/mL；With the meter of sgRNA, the end of the RNP compound Concentration is 0.1-1.5 μm of ol/ μ L.

Wherein, optionally, in step S3, the condition that electricity turns includes: that electric field strength is 50-250V/cm, single pulse time For 2-15ms, the time interval between adjacent two subpulse is 10-60s, and total pulses are 2-10 times.

Wherein, optionally, it in step S4, after electricity turns in 5-20 minutes, is added in the material after turning to the electricity Culture of the suspension of AAV virion to be transfected to carry out transfection in 8-24 hours, after being transfected.

Wherein, optionally, in step S4, the additional amount of the suspension of AAV virion to be transfected makes AAV to be transfected The MOI value of virion is 10⁴-10⁶.The ratio of virus and cell quantity when MOI value is infection.

Wherein it is preferred to which the mescenchymal stem cell is placenta mesenchyma stem cell, mesenchymal stem cell, fat Mescenchymal stem cell, umbilical cord mesenchymal stem cells, palace blood mescenchymal stem cell and dental pulp mescenchymal stem cell.The AAV virus Serotype be AAV-6 virus, AAV-1 virus or AAV9 virus, the serotype of the preferably described AAV virus is AAV9 viral, Under the preferable case, the efficiency of large fragment gene knock-in stem cell can be further increased.

Wherein, the self cleavage 2A peptide, can for cutting the albumen of hepatic lineage related gene and fluorescin open Think at least one of T2A peptide, F2A peptide and P2A peptide；The selection of the fluorescent protein report gene can be wider, such as can Think at least one of EGFP, ECFP, EYFP, GFP, RFP, mCherry, tdTomato and Venus.

Wherein, optionally, the sequence of the fluorescent protein report gene is as shown in SEQ ID NO.9；The bone of the carrier Frame sequence is as shown in SEQ ID NO.10.The frame sequence of the carrier refers to the sequence for being not inserted into the carrier of template DNA.

Present invention be described in more detail by the following examples:

Embodiment 1

Construct the cell membrane of the insertion EGFP reporter gene in the Serum Albumin Gene (ALB) of placenta mesenchyma stem cell Type.Using ALB gene original position gene promoter, P2A-EGFP is inserted into before terminator codon.

1, sgRNA design and synthesis

(1) the 14th extra of ALB gene is directed to by online sgRNA design tool (http://crispr.mit.edu/) The ALB sgRNA of targets identification is designed on aobvious son.ALB sgRNA:5'- aaugugauguuauaagccuaguuuuagagcua gaaauagcaaguuaaaauaaggcuaguccguuaucaa cuugaaaaaguggcaccgagucggugcuuuuuuu--3’( SEQ ID NO.1).Wherein 1-20 sequences are identification motif, remaining sequence is tracrRNA.

(2) sgRNA is synthesized and by Integrated Dna Technologies.USA company in the sgRNA sequence Repairing for O-Me, phosphorothioate is added respectively on No. two positions of three bases at 5 ' ends and 3 ' end ends and third place Decorations.

2, the activity of in vitro method detection sgRNA

(1) genome amplification identification target sequence segment (2000bp), primer are limited by giving birth to work bioengineering (Shanghai) share Company's synthesis.

ALB FW:5 '-gagtctatttgtagaaaatg-3 ' (SEQ ID NO.7),

ALB REV:5 '-ctctactgaagcgactggag-3 ' (SEQ ID NO.8).

(2) pcr amplification product 200ng, sgRNA 100ng, SpCas9 200ng (are purchased from Sigma-Aldrich, goods Number: TGEN-CP-500UG), 10 × buffer, 2 μ L is configured to 20 μ L reaction systems.Reaction system keeps the temperature one hour at 37 degree, One hour is kept the temperature at 70 degree.

(3) using TAE configuration 1% BioWest agarose gel, the electrophoresis 30min under the voltage of 90V, using gel at As instrument observes result.

3, the selection of AAV packaging system and plasmid construction

(1) cell to be edited is people's placenta mesenchyma stem cell, according to the AAV tissue affinity table of comparisons, preferably serotype For the packaging system of AAV-9.

(2) the overall package capacity of AAV-9 is 4.7Kb.The segment being inserted into the carrier loaded of AAV packaging system should wrap Containing left homology arm (500bp or so), Insert Fragment and right homology arm (500bp or so).

Left homology arm sequence is SEQ ID NO.2, wherein 490-492 bit base is the site PAM by mutation.

Right homology arm sequence is SEQ ID NO.3.

Insert Fragment is P2A and EGFP, and total 774bp, sequence is as shown in SEQ ID NO.9.Using it is seamless clone (High-fidelity DNA assemble kit, be purchased from NEB (Beijing) Co., Ltd, article No.: E2621S) method will Cassette is inserted on pAAV carrier, and pAAV carrier sequence is SEQ ID NO.10.

4, AAV virus packaging and purifying

(1) virus packaging on the day before by HEK293T cell according to every ware 5 × 10⁶Quantity plant to diameter 10cm's Containing 10mL complete medium (DMEM+10%FBS+1%P/S is dual anti-), (DMEM culture medium, is purchased from ThermoFisher Scientific, Inc., article No.: C11995500BT；FBS, is purchased from ThermoFisher Scientific, Inc., article No.: sv30087.02；P/S is dual anti-, is purchased from ThermoFisher Scientific, Inc., article No.: SV30010) CORNING training It supports in ware, plants 30 wares altogether.In 37 degree, 5%CO₂Cell incubator in cultivate 24 hours.

(2) the virus packaging same day checks whether HEK293T cell confluency degree reaches 80%.Every ware is independent according to the following steps It configures rotaring redyeing system: by the pAAV-Cassette of 10 μ g, using nothing after the pAAV9-RC mixing of the pHelper of 10 μ g, 10 μ g It is mixed after the DMEM adjustment volume to 910 μ L of serum through vortex oscillator, the PEI that 90 μ L are added (is purchased from Polysciences Asia Pacific, Inc., article No.: 23966-2) after reuse vortex oscillator mixing, stand 15 minutes.By CORNING Complete medium in culture dish is substituted for 9mL serum free medium, then will be equal by the DNA-PEI compound stood before It is even to be added drop-wise in culture dish, it softly shakes and even is placed on 37 degree, 5%CO₂Cell incubator in cultivate 6 hours.It will after transfection 6h Serum free medium in CORNING culture dish is substituted for complete medium, in 37 degree, 5%CO₂Cell incubator in continue Culture.

(3) after transfection 60 hours, supernatant cell is harvested respectively.

Obtained supernatant will be collected and discard impurity after ten minutes through 4 degree of centrifugations of 4000rpm.It will go on deimpurity to reset and add Enter Amicon Ultra-15 and surpass and (be purchased from Merck Chemical Engineering Technology (Shanghai) Co., Ltd., article No.: UFC 905096) from column, passes through Volume concentration to 10 was arrived into 15mL in centrifugation 30 minutes after 4000rpm several times, 4 degree.The HEK293T scraped with cell scraper is thin Born of the same parents blow even and are transferred in 50mL centrifuge tube with appropriate culture medium, abandon supernatant, Suo Youchen after 1500rpm, 4 degree of centrifugation 10min It forms sediment in total plus 3mL cell lysis buffer solution (150mM NaCl, 20mM tris pH8.0) makes its resuspension.Cell will be resuspended -80 Multigelation is three times in DEG C alcohol bath and 37 DEG C of water-baths.The cell suspension of the supernatant freeze thawing of concentration is mixed, 1M is added MgCl₂To final concentration of 1mM.Addition Benzonase (is purchased from Merck Chemical Engineering Technology (Shanghai) Co., Ltd., article No.: 70746- 1kU) to final concentration of 25U/mL, 37 DEG C of reaction 40min after mixing.50mL centrifuge tube is taken out, 4 DEG C, 4000rpm is centrifuged 20min, Take supernatant.

(4) Sigma-Aldrich (is purchased from, article No.: D1556- using Iodixanol density-gradient centrifugation method purified virus 250mL).Configure Iodixanol gradient 17%:5mL 10 × PBS, 0.05mL 1M MgCl₂,0.125mL 1M KCl,10mL 5M NaCl, 12.5mL Optiprep, adds water to supply 50mL.25%:5mL 10 × PBS, 0.05mL 1M MgCl₂, 0.125mL 1M KCl, 20mL Optiprep, 0.1mL 0.5%phenol red, adds water to supply 50mL.40%:5mL 10 ×PBS, 0.05mL 1M MgCl₂, 0.125mL 1M KCl, 33.3mL Optiprep adds water to supply 50mL.60%: 0.05mL 1M MgCl₂, 0.5% phenol red of 0.125mL 1M KCl, 50mL Optiprep, 0.025mL.To hypervelocity The iodine gram sand of 3.5mL 60%, 3.5mL 40%, 4mL 25%, 4mL 17% are successively slowly added in centrifuge tube from the bottom to top Alcohol.The supernatant cell pyrolysis liquid of concentration is slowly added in centrifuge tube top layer, fills centrifuge tube with cell lysis buffer solution.Make Angle rotor is determined with Beckman L-80XP landing ultracentrifuge, 70Ti, is accelerated 6, is slowed down 4 degree of 9,60000rpm and be centrifuged 2 hours. 40% concentration layer Iodixanol is drawn with tack syringe, Amicon Ultra-15 is transferred to and surpasses from column, 10mL is added 4 DEG C of PBS 4000rpm are centrifuged 20 minutes, are repeated 3 times.By viral centrifugal concentrating to 1mL.

(5) titre detection is carried out to AAV9 using qPCR.According to EGFP primers, make the length of qPCR product about 200bp.QPCR primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

AAVGFPF:5’-tcagcttcaggcaccaccac-3’(SEQ ID NO.11)。

AAVGFPR:5’-tgaacttgtggccgtttacgtcg-3’(SEQ ID NO.12)。

7 AAV9 recombinant plasmid standard items are prepared, gradient dilution is carried out with the ratio of 1:10.It is below dense from 10ng/ μ L Degree start to dilute, respectively 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L and 0.00001ng/μL。

Diluted DNA copy number is calculated according to the following formula:

Virus Sample is pre-processed using DNase (it is purchased from precious bioengineering (Dalian) Co., Ltd, article No.: 2270A).(Japan is purchased from and spins (Shanghai) Biotechnology Co., Ltd, article No.: QPS-201) configuration using 2 × SYBR PCR mix QPCR reaction system.Use Roche480II real-time fluorescence quantitative PCR system carries out quantitative PCR.According to Ct value draws standard curve, and calculates the titre of AAV9.

5, the assembling of RNP compound and the pretreatment of cell to be edited

(1) by SpCas9 (300 μ g/ml of final concentration) and ALB sgRNA (175 μ g/ml of final concentration) according to mole of 1:3 Than carrying out being blended in incubation at room temperature 10min, to form SpCas9/sgRNA RNP compound.

(2) observation placenta mesenchyma stem cell, which converges rate and reaches 80%, moves back except the DMEM/F12 culture medium containing 10%FBS (it is purchased from ThermoFisher Scientific, article No.: 11320082) Inc., is rinsed primary with PBS.0.05% pancreatin is added (being purchased from ThermoFisher Scientific, Inc., article No.: 25200-072) is allowed to that ware bottom is completely covered.37 DEG C of incubation 3-5 Stop digestion after minute, sucks pancreatin.At once fresh DMEM/F12 complete medium is added, is blown and beaten and is cultivated with 1mL rifle sector Ware bottom, the stem cell colonies for attaching ware/bottom of bottle fall off, and soft slowly pressure-vaccum mixes, and stem cell suspension is made.Use hemocytometer Number plate counts cell density.It (is purchased from using Opti-MEM (magnesium chloride of ATP, 23.6mM of 14.5mM) Article No.: 11058021) ThermoFisher Scientific, Inc. adjust cell density to 5 × 10⁷/mL。

(3) by 10 μ L by incubation at room temperature SpCas9/sgRNA SNP compound (with the meter of sgRNA, the RNP The content of compound is 7.5 μm of ol) and 10 μ L by counting and using the placenta mesenchyma stem cell suspension of Opti-MEM resuspension (wherein cell quantity is 5 × 10⁵It is a) it mixes, 20 μ L mixed liquors are transferred in 16 hole electricity rotating plate items.Use Lonza 4D (electric field strength 150V/cm, the single pulse time is 10ms to nuclear transfection Systematic selection CB150 mode, between adjacent two subpulse Time interval be 20s, total pulses be 5 times) carry out electricity turn.Cell is transferred to containing DMEM/F12 by electricity at once after the completion of turning In 37 degree, 5%CO in 24 orifice plates of complete medium₂Cell incubator in continue to cultivate.

(4) electricity has turned to start in the 5 to 20th minute according to 1 × 10⁵MOI value it is soft must be added dropwise AAV9, and turned in electricity It is added dropwise in 20 minutes.

(5) electricity removes old culture medium after having turned 24 hours, is changed to DMEM/F12 complete medium.

6, the culture of monoclonal cell strain:

(1) electricity has turned to rinse a culture dish using PBS on the 48th hour.Pancreatin is added to be allowed to that ware bottom is completely covered.37℃ Stop digestion after being incubated for 3-5 minutes, sucks pancreatin.At once fresh DMEM/F12 complete medium is added, it is fan-shaped with 1mL rifle Culture dish/bottom of bottle is blown and beaten, the stem cell colonies for attaching ware bottom fall off, and soft slowly pressure-vaccum mixes, and guarantee that iuntercellular does not glue Even, stem cell suspension is made.Cell density is counted using blood counting chamber.It is thin by 15000, each 10cm culture dish The ratio of born of the same parents in the 10cm CORNING culture dish of cell seeding to the complete medium containing DMEM/F12 in 37 degree, 5%CO₂ Cell incubator in continue to cultivate.

(2) it is observed and is removed old culture medium within every 24 hours, be changed to new DMEM/F12 complete medium.72 It can be in microscopic observation to the formation of clone after hour.

(3) 12 to 14 days sizes that can see clone under ten times of object lens have been equivalent to a coin after electricity turns. Clone cannot be allowed to continue to become larger or intersect.120 μ L DMEM/F12 complete mediums are added in each hole on 96 orifice plates, Marking plate O.It is observed in super-clean bench by microscope, P200 pipettor is adjusted to 45 μ L and uses the pipette tips with filter core Broken clone is scraped, collect cell with pipettor and is transferred in the aperture of 96 orifice plates.

(4) old culture medium is removed after every 24 hours after clone's picking, is changed to new DMEM/F12 and cultivates completely Base, cell confluency rate reaches 80% after four days.

(5) 133 μ L DMEM/F12 complete mediums are added in each hole on two piece of 96 orifice plate, are respectively labeled as plate A, plate B.Every hole of plate O is rinsed using the PBS of 150 μ L.The pancreatin of 35 μ L, digestion 5 to every hole after ten minutes are added in every hole The DMEM/F12 complete medium of 165 μ L of middle addition.The cell suspension for pipetting 66 μ L respectively from hole is transferred to plate A's and plate B In respective apertures.Plate A is extracted for genome, and plate B is spare.The cells frozen storing liquid of 165 μ L is directly added in each hole of plate O It is stored afterwards as profound hypothermia refrigerator.

Embodiment 2

The thin of EGFP reporter gene is inserted into building in placenta mesenchyma stem cell, alpha fetoprotein (AFP) gene (AFP) Born of the same parents' model.Using AFP gene original position gene promoter, P2A-EGFP is inserted into before terminator codon.Because of two editings of AFP Variant variant possesses identical CTD so being marked to two kinds of editing variants of AFP simultaneously in the present embodiment.

By online sgRNA design tool (http://crispr.mit.edu/) in 51 exon of COL1A1 gene The sgRNA of targets identification is designed in the sequence within terminator codon (TAA) upstream 100bp.

AFP sgRNA:5’-gguuguagaccccaaaaguaguuuuagagcuagaaauagcaaguuaaaau aagg cuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’。(SEQ ID NO.4)。

Wherein 1-20 sequences are identification motif, remaining sequence is tracrRNA.

The sgRNA is synthesized by Integrated Dna Technologies.USA company and at the 5 ' end and 3 ' ends of sequence The modification of addition -2-O-Me, -3-phosp horothioate respectively on No. two positions of three bases at end and third place.

2, the activity of in vitro method detection sgRNA

(1) target sequence segment (2000bp) is identified from genome amplification, primer is had by giving birth to work bioengineering (Shanghai) share The synthesis of limit company.

AFP FW:5 '-gagtctatttgtagaaaatg-3 ', (SEQ ID NO.13),

AFP REV:5 '-ctctactgaagcgactggag-3 ', (SEQ ID NO.14).

(2) pcr amplification product 200ng, sgRNA 100ng, SpCas9 200ng (are purchased from Sigma-Aldrich, goods Number: TGEN-CP-500UG), 10 × buffer, 2 μ l is configured to 20 μ l reaction systems.Reaction system keeps the temperature one hour at 37 degree, 70 degree keep the temperature one hour.

(3) using TAE configuration 1% BioWest agarose gel, the electrophoresis 30min under the voltage of 90V, using gel at As instrument observes result.

3, the selection of AAV packaging system and plasmid construction

(1) cell to be edited is placenta mesenchyma stem cell strain, according to the AAV tissue affinity table of comparisons, preferably serotype For the packaging system of AAV9.

(2) the overall package capacity of AAV9 is 4.7Kb.The segment being inserted into the carrier loaded of AAV packaging system should wrap Containing left homology arm (500bp or so), Insert Fragment and right homology arm (500b p or so).Left homology arm sequence is SEQ ID NO.5, wherein 493-495 bit base is the site pam by mutation), right homology arm sequence is SEQ ID NO.6.It is inserted into piece Section is P2A and E GFP, and total 774bp, sequence is as shown in SEQ ID NO.9.Using it is seamless clone (High-fidelity DNA assemble kit, be purchased from NEB (Beijing) Co., Ltd) method Cassett e is inserted on pAAV carrier, carrier sequence It is classified as SEQ ID NO.25.In order to avoid SpCas9/sg RNA RNP compound influences the secondary cut of recombination site The efficiency of gene editing (is purchased from precious bioengineering (Dalian) Co., Ltd, goods by Pr imerStar high-fidelity DNA polymerase Number: point mutation (CGG sports CTG) R 044A) is introduced in the site PAM using the method that PCR carves ring, so as to avoid Cutting of the SpCas9/sgRNA RNP compound to recognition site.Point mutation primer is limited by giving birth to work bioengineering (Shanghai) share Company's synthesis.

AFP-PAM-mut-FW:5’-aaactcgtgctgctttaggagtttaaattactt-3’(SEQ ID NO. 15)。

AFP-PAM-mut-Rev:5’-aagtaatttaaactcctaaagcagcacgagttt-3’(SEQ ID NO. 16)。

4, AAV virus packaging and purifying

With embodiment 1

5, the assembling of RNP compound and the pretreatment of cell to be edited

With embodiment 1

6, Lonza 4D system electricity turns and AAV infects

With embodiment 1

7, the culture of monoclonal cell strain

With embodiment 1

Comparative example 1

It carries out according to the method for embodiment 1, the difference is that electricity has turned 35min and started according to 1 × 10⁵MOI gently AAV9 is added dropwise, and has turned to be added dropwise in 50 minutes in electricity.

Testing example 1

1, fluorescence detection allele is inserted into Efficiency testing

Test sample: embodiment 1,2 and comparative example 1 by electricity turn and AAV infection after the 4th day after pancreatin digests Fresh DMEM/F12 complete medium, which is added, makes it that manufactured stem cell suspension be resuspended.

Detection method: because the P2A-EGFP sequence expressing green fluorescent protein being inserted on target gene seat, uses stream Formula cell instrument detects and counts the cell quantity that can issue green fluorescence and cell total amount.The cell of green fluorescence will be issued Quantity divided by cell total amount can with arrive total editorial efficiency.

Experimental result: 1 editorial efficiency of embodiment is 78.1%, and 2 editorial efficiency of embodiment is 76.6%, and comparative example 1 is edited Efficiency is 1.2%.

2, PCR detects allele and is inserted into efficiency

Test sample: the monoclonal cell strain (A plate and B plate) of 96 orifice plate cultures

Detection method: the design primer outside homology arm.Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

Embodiment 1, comparative example 1 use ALB-FW, ALB-REV.Embodiment 2 uses AFP-FW, AFP-REV.

Clone's genome obtained in 96 orifice plates is subjected to PCR amplification.PCR product is subjected to electrophoresis and is analyzed.It is real Applying example 1, comparative example 1 only 2000bp or so band is non-editor's cell, and only 2800bp is that diallele editor is thin Born of the same parents, it is monoallelic editor's cell that existing 2000bp or so band, which also has 2800bp,.It marks respectively and counts these three types of volumes Collect the ratio of type.There was only 2000bp or so band in embodiment 2 is non-editor's cell, and only 2800bp is double equipotential bases Because editing cell, it is monoallelic editor's cell that existing 2000bp or so band, which also has 2800bp,.It marks and counts respectively The ratio of these three types of editing types.

Experimental result: 1 editorial efficiency of embodiment: allele double editors 30.2%, allele list editor 55.3%, it is right 1 editorial efficiency of ratio: allele double editors 0.9%, allele list editor 1.5%；2 editorial efficiency of embodiment: equipotential base Because of double editors 23.4%, allele list editor 58.8%；It can be seen that electricity turns the time interval with virus transfection to gene knock-in Efficiency is affected, and preferably after electricity turns in 5-20 minutes, AAV to be transfected is added in the material after turning to the electricity In the case that the suspension of virion is to carry out transfection in 8-24 hours, the efficiency highest of gene knock-in.

3, break up under constructed cell model induces in vitro to mature hepatic lineage and detect

Referring to Tal é ns-Visconti R in article Human mesenchymal stem cells from adipose Method in tissue:Differentiation into hepatic lineage. knocks in EGFP in ALB in embodiment 1 Placenta mesenchyma stem cell and embodiment 2 in carried out on the placenta mesenchyma stem cell that AFP knocks in EGFP induction make it It is divided into mature hepatic lineage.During mature using the 4th induced medium Cell differentiation inducing activity (total induction the 13rd, 15,17,19,21 days) when carry out immunocytochemical stain after the cells are fixed (primary antibody of ALB be Anti-Albumin antibody Purchase Ai Bokang (Shanghai) trade Co., Ltd, article No.: ab106582, corresponding fluorescence secondary antibody are Goat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor 555 is purchased from ThermoFisher Scientific, Inc., Article No.: A-21424；The primary antibody of AFP are as follows: 1 Fetoprotein antibody [AFP-01] of Anti-alpha purchases Ai Bokang (Shanghai) trade Easy Co., Ltd, article No.: ab3980, corresponding fluorescence secondary antibody are Goat anti-Mouse IgG (H+L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor Plus 555 is purchased from ThermoFisher Scientific, Inc., article No.: A-21424).Fluorescence microscope green fluorescence channel and red fluorescence is used to lead to respectively in two groups of cells Road will count the coincidence factor of green fluorescence and red fluorescence observation after figure layer merge (coincidence).

Cell hepatoid differentiation state-detection in 1 embodiment 1 of table

Cell hepatoid differentiation state-detection in 2 embodiment 2 of table

From in the statistical data of Tables 1 and 2 as can be seen that ALB and reality in cell and embodiment 1 indicated by EGFP It is consistent to apply cell indicated by AFP in example 2.That is in the EGFP label established by embodiment 1 and embodiment 2 Placenta mesenchyma stem cell can can be with the degree of its hepatoid differentiation of real-time detection with the degree of real-time detection its hepatoid differentiation It can be with the degree of its hepatoid differentiation of real-time detection.

The preferred embodiment of the disclosure is described in detail above, still, during the disclosure is not limited to the above embodiment Detail a variety of simple variants can be carried out to the technical solution of the disclosure in the range of the technology design of the disclosure, this A little simple variants belong to the protection scope of the disclosure.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the disclosure to it is various can No further explanation will be given for the combination of energy.

In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally Disclosed thought equally should be considered as disclosure disclosure of that.

Sequence table

<110>Hangzhou Guan Zi health Science and Technology Ltd.

<120>a kind of prepare can the method for cell model broken up to mature hepatic lineage of real-time detection mescenchymal stem cell

<130> 10971-K-HZGZ

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 103

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 1

aaugugaugu uauaagccua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 2

<211> 500

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

actttgtttt tatctcctgc tctattgtgc catactgtta aatgtttata atgcctgttc 60

tgtttccaaa tttgtgatgc ttatgaatat taataggaat atttgtaagg cctgaaatat 120

tttgatcatg aaatcaaaac attaatttat ttaaacattt acttgaaatg tggtggtttg 180

tgatttagtt gattttatag gctagtggga gaatttacat tcaaatgtct aaatcactta 240

aaattgccct ttatggcctg acagtaactt ttttttattc atttggggac aactatgtcc 300

gtgagcttcc gtccagagat tatagtagta aattgtaatt aaaggatatg atgcacgtga 360

aatcactttg caatcatcaa tagcttcata aatgttaatt ttgtatccta atagtaatgc 420

taatattttc ctaacatctg tcatgtcttt gtgttcaggg taaaaaactt gttgctgcaa 480

gtcaagctgc cttaggctta 500

<210> 3

<211> 500

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

taacatcaca tttaaaagca tctcaggtaa ctatattttg aattttttaa aaaagtaact 60

ataatagtta ttattaaaat agcaaagatt gaccatttcc aagagccata tagaccagca 120

ccgaccacta ttctaaacta tttatgtatg taaatattag cttttaaaat tctcaaaata 180

gttgctgagt tgggaaccac tattatttct attttgtaga tgagaaaatg aagataaaca 240

tcaaagcata gattaagtaa ttttccaaag ggtcaaaatt caaaattgaa accaaagttt 300

cagtgttgcc cattgtcctg ttctgactta tatgatgcgg tacacagagc catccaagta 360

agtgatggct cagcagtgga atactctggg aattaggctg aaccacatga aagagtgctt 420

tatagggcaa aaacagttga atatcagtga tttcacatgg ttcaacctaa tagttcaact 480

catcctttcc attggagaat 500

<210> 4

<211> 103

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gguuguagac cccaaaagua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 5

<211> 500

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tgggatcagg tagggtggga catagataac caaattagat aaaactggtg aaacagattt 60

gatgtgaagc atttctgaaa aacatgacac aagaagatta atgttctcta atctgagaag 120

acatttattt agatatagag aacatgaaca aatagtagca gtgctttatc tgcaaacctt 180

ttaatttcta ataacttgta atttgtagag gaaggggaaa gattgagaat acgcattgat 240

ttggagattg ttatagaaga aaactgttga tgtgaaagaa tattgttttc tccctggctt 300

ttactatccc aggttgttgg catcagagat gtgtttcttc atttttaact tagttaatct 360

acaaacctat gaattcaccc cggattgtag agtgttaact gtatgattgg tataataatc 420

catttcttta tctgattatg tttattctta attttcaggg acaaaaactg atttcaaaaa 480

ctcgtgctgc tttaggagtt 500

<210> 6

<211> 500

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

taaattactt caggtaacaa aacattcaga caagcctgaa tacaatgttg tttctccaga 60

aatatcaatc cataatgaga tagatcatga ggagtgccat taattctctt aaaaatacat 120

ggaattcaaa aaaaagttta ttttaaaaac acttgaacaa aattacgcac acaatgttaa 180

attagtggct caactatgca aaatcctttt tggttattta aaagacttca acaaatgcta 240

tcagaagact ttcctacgta tccaatattt ctctgatata aaataataga accagttact 300

tactgcacct attagtttaa ttagtattta atatattttt gctcatattg caggggaaga 360

gaagacaaaa cgagtctttc attcggtgtg aacttttctc tttaatttta actgatttaa 420

cactttttgt gaattaatga aatgataaag acttttatgt gagatttcct tatcacagaa 480

ataaaatatc tccaaatgtt 500

<210> 7

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gagtctattt gtagaaaatg 20

<210> 8

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ctctactgaa gcgactggag 20

<210> 9

<211> 774

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc 60

aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 120

aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 180

accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 240

accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 300

ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 360

gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 420

atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 480

tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 540

gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 600

cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 660

acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 720

ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga attc 774

<210> 10

<211> 2949

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgcg aatgctgcgg cgcgcggcta gcctgatttt 180

gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact 240

ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg 300

ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg 360

atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac 420

catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 480

tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 540

tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 600

gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta 660

gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 720

atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg 780

atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 840

aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta 900

caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 960

cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 1020

ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 1080

tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 1140

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 1200

caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 1260

ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 1320

gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 1380

tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 1440

ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 1500

tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 1560

atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 1620

gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 1680

caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 1740

ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 1800

ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 1860

ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 1920

ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 1980

gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 2040

ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 2100

taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 2160

agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 2220

atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 2280

aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 2340

caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 2400

ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 2460

cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 2520

tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 2580

gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 2640

ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 2700

gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 2760

caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 2820

ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 2880

tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 2940

ctcacatgt 2949

<210> 11

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

tcagcttcag gcaccaccac 20

<210> 12

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tgaacttgtg gccgtttacg tcg 23

<210> 13

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

gagtctattt gtagaaaatg 20

<210> 14

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ctctactgaa gcgactggag 20

<210> 15

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

aaactcgtgc tgctttagga gtttaaatta ctt 33

<210> 16

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

aagtaattta aactcctaaa gcagcacgag ttt 33

Claims (10)

1. A method for preparing a cell model capable of real-time detection of mesenchymal stem cells differentiated into mature hepatic-like cells, characterized in that the method may further comprise the steps: S1, mixing the sgRNA and Cas9 protein for the knock-in site of the liver-like cell-related genes to form an RNP complex; the liver-like cell-related genes include ALB and/or AFP; the knock-in of the liver-like cell-related genes The site is between the last sense codon and the stop codon of the liver-like cell-related gene; S2. Packaging the homologous recombination vector inserted with the template DNA into the AAV virus to form AAV virus particles to be transfected; the template DNA includes the left homology arm sequence for the knock-in site of the liver-like cell-related gene and A right homology arm sequence, a self-cleaving 2A peptide coding sequence and a fluorescent protein reporter gene are inserted between the left homology arm sequence and the right homology arm sequence, so that after directional knock-in, a gene encoding A fusion gene of a fusion protein composed of related genes, 2A peptides and fluorescent proteins in series; S3. Mixing the suspension of mesenchymal stem cells and the suspension of the RNP complex and performing electroporation to obtain a culture after electroporation; S4. Within 1-30 minutes after electroporation, add the suspension of AAV virus particles to be transfected to the material after electroporation to perform transfection for 4-30 hours, and obtain a transfected culture; S5. Performing monoclonal culture after extreme dilution of the transfected culture, and screening out a monoclonal cell line that has undergone gene-directed knock-in by PCR and sequencing. 2. The method according to claim 1, wherein the sgRNA has a chemical modification group; the chemical modification group is preferably a methyl chemical modification group or a phosphorothioate chemical modification group; for liver-like cells The molar ratio of sgRNA and Cas9 protein at the knock-in site of the relevant gene is 1:1 to 1:5. 3. The method according to claim 1 or 2, wherein the time for mixing the sgRNA targeting the knock-in site of the hepatoid cell-related gene and the Cas9 protein is 5-20 minutes, and the temperature is 10-40°C. 4. The method according to claim 1, wherein, in step S3, when the suspension of the mesenchymal stem cells is mixed with the RNP complex, relative to every 10 ⁶ of the mesenchymal stem cells, sgRNA In terms of the amount of sgRNA, the amount of the RNP complex is 10-50 μmol, and in the suspension of the mesenchymal stem cells, the cell concentration is (1-5)×10 ⁷ cells/mL; in terms of the amount of sgRNA, the The final concentration of RNP complexes was 0.1-1.5 μmol/μL. 5. The method according to claim 1 or 4, wherein, in step S3, the conditions for electroporation include: the electric field strength is 50-250V/cm, the single pulse time is 2-15ms, and the time between two adjacent pulses The time interval is 10-60s, and the total number of pulses is 2-10 times. 6. The method according to claim 1, wherein, in step S4, within 5-20 minutes after electroporation, a suspension of AAV virus particles to be transfected is added to the material after electroporation to carry out 8- After 24 hours of transfection, the transfected cultures were obtained. 7. The method according to claim 1 or 6, wherein, in step S4, the amount of the suspension of AAV virus particles to be transfected is such that the MOI value of the AAV virus particles to be transfected is 10 ⁴ -10 ⁶ . 8. The method according to claim 1, wherein the mesenchymal stem cells are placental mesenchymal stem cells, bone marrow mesenchymal stem cells, fat mesenchymal stem cells, umbilical cord mesenchymal stem cells, uterine blood mesenchymal stem cells and dental pulp mesenchymal stem cells, the serotype of the AAV virus is AAV-6 virus, AAV-1 virus or AAV9 virus. 9. The method according to claim 1, wherein the self-cleaving 2A peptide is at least one of T2A peptide, F2A peptide and P2A peptide; the fluorescent protein reporter gene is EGFP, ECFP, EYFP, GFP, At least one of RFP, mCherry, tdTomato and Venus. 10. The method according to claim 1, wherein the sgRNA is as shown in SEQ ID NO.1 or 4; correspondingly, the left homology arm sequence is as shown in SEQ ID NO.2 or 5; correspondingly , the sequence of the right homology arm is shown in SEQ ID NO.3 or 6.