CN103388006B - A kind of construction process of site-directed point mutation - Google Patents

Abstract

The invention discloses a method for constructing gene site-directed mutagenesis, which determines the target sequence in the rat target genome sequence that can be recognized by the artificially modified CRISPR-Cas system; constructs a guide RNA sequence that can recognize and guide the CAS protein to the target sequence of the target gene ( guide? RNA, gRNA); the above-mentioned gRNA sequence and CAS protein coding nucleic acid sequence or gRNA sequence and in vitro expressed CAS protein mixture are introduced into rat embryonic cells. The invention does not need to construct a homologous recombination targeting vector, does not need to screen ES targeting cells, has a high proportion of mutants, is easy to operate, can realize simultaneous knockout of multiple sites, and can greatly reduce experiment costs and shorten experiment periods. <pb pnum="1" />

Description

A method for constructing gene site-directed mutation

technical field

The invention relates to the technical field of genetic engineering, in particular to a method for constructing gene site-directed mutation in rat embryonic cells.

Background technique

Rats have been used as animal models in the fields of physiology, pharmacology, toxicology, nutrition, behavior, immunology, and oncology for more than 150 years. Similar to mice, rats have the advantages of small size, short intergenerational time, multiple births per litter, and low feeding costs. At the same time, compared with mice, rats have a larger body size, which is convenient for observation and surgical operations; the diet of rats is closer to that of humans, which is convenient for nutritional research; rats are more sensitive to toxic substances and are suitable for use in medicines. Toxicology experiment. At present, each drug needs to be tested in rats for toxicology before it goes on the market. Due to inherent genetic reasons, rat disease models can more realistically reproduce the symptoms of some human diseases. Rats have a strong desire to explore the environment and relatively complex and advanced neural activities, which are suitable for neurological, cognitive and behavioral research. Among the animal models of human diseases included in PubMed, the number of rats used as models occupies the first place.

However, with the maintenance of stemness in vitro culture of mouse embryonic stem cells and the realization of gene targeting based on homologous recombination in mouse embryonic stem cells cultured in vitro in 1981, the individualized site-specific modification of mouse genes has become a routine operation. The scientific community has turned to mice to study the function of homologous human genes and to model human diseases. It was not until 2008 that the isolation and culture of rat embryonic stem cells in the true sense, as well as the successful report of germline integration and passage of chimeric rats, were reported. However, due to the complex and harsh culture conditions and the influence of resistance selection on stemness maintenance, it is still difficult to perform gene targeting in rat embryonic stem cells. Similar to that in mice, gene targeting based on homologous recombination in rat embryonic stem cells must be subject to the following limiting factors: 1. The construction process of targeting vectors is long and difficult. 2. Since the incidence of spontaneous homologous recombination in animal cells is only 10 ^-5 -10 ^-6 , after electrotransferring the targeting vector into rat embryonic stem cells, it will take a lot of time, manpower and material resources to screen the embryonic stem cells that undergo the targeted homologous recombination . 3. The culture conditions of ES cells are harsh in the process of in vitro culture. A little carelessness will cause their irreversible differentiation, and make them lose the ability of germline transfer, and cannot produce mutant progeny in subsequent operations. 4. Microinjected ES cells have only a certain probability of successfully integrating into germline cells capable of forming gametes. 5. ES cells are required to be in a good growth and undifferentiated state. 6. If there are ready-made ES cells with the target gene targeted, but different from the rat strain to be studied, it will take a lot of time for backcrossing to dilute the original genetic background of the ES cells.

The artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology developed in recent years has solved the above problems to a certain extent. ZFN is a recombinant protein composed of a zinc finger protein targeting a specific DNA sequence and a FokI endonuclease cleavage domain with non-specific effects. After a pair of artificially designed ZFNs that recognize a specific DNA sequence bind to the target DNA sequence (usually two adjacent segments of 18-24bp each, with an interval of 4-7bp), their FokI endonuclease will form a dimer, Thereby cutting the double strand of DNA, forming a double strand break (double strand break, DSB). The damage is mainly repaired by error-prone non-homologous end joining (Non-homologous end joining, NHEJ) and high-fidelity homologous recombination (homologous recombination, HR). Among them, non-homologous end joining can be used to knock out the target gene by causing a frameshift of the target gene. By microinjecting ZFN-encoding DNA or corresponding mRNA at the fertilized egg stage, gene knockout in rats, mice, zebrafish and other model organisms has been achieved.

Although ZFN technology has broad prospects in basic research and application fields, the construction of ZFNs with high specificity and affinity for specific DNA sequences according to needs requires a large workload, long cycle, and low success rate, which have become obstacles to the large-scale application of ZFNs. bottleneck. The DNA recognition domain of ZFN is composed of several zinc finger modules, and each zinc finger module recognizes a specific 3 consecutive bases, so adjacent 3 to 6 zinc finger modules can recognize consecutive 9 to 18 bp bases on DNA base. This triplet recognition pattern means less flexibility in the choice of DNA sequences to target. Although there are hundreds of zinc finger modules, not all possible base combinations are covered. At the same time, because the interaction between adjacent zinc finger modules will affect the specificity (context dependence) of its base recognition, ZFN needs to undergo a lot of optimization during the construction process to achieve specific binding to the target gene. In addition, the relatively low DNA binding specificity of ZFNs makes the overexpression of ZFNs in cells cause high cytotoxicity due to off-target effects.

Transcription Activator-Like Effector Nuclease (TALEN) is another targeted genome editing technology after ZFN. Similar to ZFN, this technology combines TAL effectors targeting specific DNA sequences with FokI nuclease to form a fusion protein. After a pair of artificially designed TALENs that recognize a specific DNA sequence bind to the target DNA sequence (usually 15-20 bp between two adjacent segments, with an interval of 15-20 bp), their FokI endonuclease will form a dimer, Thereby cutting the double strand of DNA, forming a double strand break (double strand break, DSB). The damage is mainly repaired by error-prone non-homologous end joining (Non-homologous end joining, NHEJ) and high-fidelity homologous recombination (homologous recombination, HR). Among them, non-homologous end joining can be used to knock out the target gene by causing a frameshift of the target gene. By microinjection of ZFN-encoding DNA or corresponding mRNA at the fertilized egg stage, gene knockout in rats, rats, zebrafish and other model organisms has been achieved.

Different from ZFN, TALE (TAL effectors) are composed of 12 or more serial "protein modules" that specifically recognize DNA and the N-terminal and C-terminal sequences on both sides. The DNA recognition module of TAL effectors consists of 34 amino acids. The specificity of the DNA recognition module of each TAL effectors to bind to DNA is determined by the 12th and 13th amino acid residues, which are called repeat variable di- Residues (RVDs) sites. RVDs have a constant correspondence with the four bases A, G, C, and T, that is, NI recognizes A, NG recognizes T, HD recognizes C, and NN recognizes G. Therefore, in order for TAL effectors to recognize and bind to a specific nucleic acid sequence, it is only necessary to serially clone the DNA recognition modules of TAL effectors according to the target DNA sequence. In actual operation, two adjacent (15-20 bases apart) target sequences (generally more than a dozen bases) are selected in the target gene to construct TAL effectors recognition modules respectively.

Compared with ZFN, TALEN's DNA recognition module has a clear one-to-one correspondence with a single base, and the arrangement and combination of modules does not affect each other's DNA recognition and binding capabilities, thus successfully solving the problem that conventional ZFN methods cannot recognize any target DNA sequence. As well as the problem that the recognition characteristics are often affected by the upstream and downstream sequences, the construction of TALEN's tandem DNA recognition modules eliminates the corresponding complex and expensive optimization and screening process in the construction of ZFNs, which is convenient for conventional laboratories to construct targeted targets by themselves. Gene TALEN.

However, in the construction process of TALEN, about 20 highly repetitive small fragments of DNA need to be assembled into a size of about 2kb. The process is still time-consuming and cumbersome, and there are certain unstable factors. In addition, because TALEN requires the 5' end of the target to be adjacent to the site, and the base of the 3' end of the target itself must be T, plus the requirement for the appropriate length of the TALEN target itself and the spacer (Spacer) (15-20bp), in practice The frequency of ideal target sites in the operation is low, which limits the flexibility of targeting site selection to a certain extent.

A mechanism of adaptive immunity against foreign DNA fragments such as phages and plasmids has recently been elucidated in bacteria and archaea. The system consists of Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes. The immune interference process of the CRISPR system mainly includes three stages: adaptation, expression, and interference. During the adaptation phase, the CRISPR system integrates a short piece of DNA from a phage or plasmid between the leader sequence and the first repeat, with each integration being accompanied by duplication of the repeat to form a new repeat-spacer unit . During the expression phase, the CRISPR locus is transcribed into a CRISPR RNA (crRNA) precursor (pre-crRNA), which is further processed into a small crRNA at the repetitive sequence in the presence of the Cas protein and tracrRNA. Mature crRNA forms Cas/crRNA complex with Cas protein. In the interference stage, crRNA guides the Cas/crRNA complex to find the target through its complementary region to the target sequence, and at the target position, the nuclease activity of the Cas protein causes double-strand DNA breaks at the target position, thereby losing the target DNA. original function. The 3 bases immediately adjacent to the 3' end of the target must be in the form of 5'-NGG-3', thus forming the PAM (protospacer adjacent motif) structure required for the Cas/crRNA complex to recognize the target.

The Clustered regularly interspaced short palindromic repeats (CRISPR) system is divided into three families of type I, II, and III, and the type II system only needs Cas9 protein to transcode the small RNA (trans-encodedsmall RNA, tracrRNA) Pre-crRNA is processed into mature crRNA bound to tracrRNA. As a result, it has received more attention from scientific researchers and has been the most fully studied. It has been found that by artificially constructing a single-stranded chimeric guide RNA (guide RNA) that simulates the crRNA:tracrRNA complex, it can effectively mediate the recognition and cleavage of the target by the Cas9 protein, thereby providing a basis for the use of the CRISPR system in the target species. DNA modification offers broad prospects.

Contents of the invention

The invention provides a method for constructing gene site-directed mutation, comprising the following steps:

(1) Determine the target sequence targeted by the CRISPR-Cas system in the rat target genome sequence;

(2) Design and construct a gRNA nucleic acid sequence that can recognize and guide the CAS protein to the target gene target sequence;

(3) introducing the gRNA nucleic acid sequence and the nucleic acid sequence or protein encoding the CAS protein into the rat embryo cells.

Specifically, in the above step (1), the target DNA sequence is obtained from NCBI, and the range interval of the target sequence is selected; in this interval, press "GGNNNNNNNNNNNNNNNNNNNGG" (the first 20 bases are used to complement the non-coding strand of the target DNA pairing) to find the sequence that meets the targeting conditions in the non-template strand; you can also search for "CCNNNNNNNNNNNNNNNNNNNNNNCC" in the reverse complementary sequence of the candidate targeting region to find potential targets.

In the above step (2), the gRNA nucleic acid sequence can specifically bind to the CAS protein and target it to a target sequence. That is, the guide RNA in vitro transcription template consists of the T7 promoter sequence, the target-specific sequence, and the gRNA backbone part in sequence from the 5' to the 3' end. Design the corresponding guide RNA according to the target sequence information.

In the above step (3), the gRNA nucleic acid sequence and the encoded CAS protein nucleic acid sequence are respectively transcribed into RNA in vitro and purified, or, the gRNA nucleic acid sequence is transcribed into RNA in vitro and then combined with the CAS protein expressed in vitro Egg whites mixed. That is, the gRNA and the CAS protein-encoding nucleic acid sequence are transcribed into RNA in vitro, and the two are mixed in a certain ratio before microinjection; or, the gRNA is transcribed into RNA, and the in vitro expressed and purified CAS protein is mixed in a certain ratio, Then used for microinjection.

In the present invention, the CRISPR-Cas system refers to a CRISPR-Cas system suitable for artificial transformation, a nuclease system derived from an archaeal type II (CRISPR)-CRISPR-associated protein (Cas) system, which is comparable to ZFN and TALEN Compared with the system, the system is simpler and more convenient to operate.

In the present invention, the rat target genome sequence includes genome sequence of coding gene, low repetitive sequence, moderate repetitive sequence, high repetitive sequence, low copy sequence, transgenic active sequence or non-transcribed sequence between genes.

In the present invention, the rat embryo cells include rat single-cell fertilized eggs or multi-cell rat embryos. For example, rat unicellular fertilized eggs cultured in vitro, or multicellular rat embryos cultured in vitro.

In the present invention, the step (3) is to introduce the guide RNA into rat embryonic cells by microinjection. The guide RNA can be obtained by artificial synthesis, in vitro transcription of a plasmid or PCR product containing the target sequence, and the like.

In the present invention, in the step (3), the DNA break site formed by cutting the target genome is repaired by non-homologous end joining or homologous recombination.

In the present invention, the target gene mutation includes base insertion, deletion, alteration, frameshift mutation or knockout.

The invention overcomes the disadvantages of the existing technology in the construction of gene knockout rats, such as long period, heavy workload, high difficulty, and low efficiency, and at the same time eliminates the need to construct and optimize the combination of ZFN and TALEN serial base recognition modules. process, and the present invention can provide targeting sequences with a higher frequency of occurrence relative to ZFNs or TALENs, thereby providing more options for precise genome editing. The rapid and efficient construction method of gene site-directed mutation of the present invention realizes rapid construction of gene knockout rats by microinjecting in vitro transcribed guide RNA (guide RNA) nucleic acid sequences and CAS protein coding nucleic acid sequences into fertilized rat eggs. The method of the present invention utilizes RNA-guided nucleases to perform rapid gene editing, rapidly targets and changes rat genome DNA rats, and realizes rapid and efficient gene site-directed mutation. The invention does not need to construct a homologous recombination targeting vector, does not need to screen ES targeting cells, has a high proportion of mutants, is easy to operate, can realize simultaneous knockout of multiple sites, and can greatly reduce experiment costs and shorten experiment periods.

In the present invention, rat embryonic cells refer to rat embryonic cells cultured in vitro. The rat embryonic cells obtained in the present invention are used for in vitro culture or directly implanted into mother mice. The CAS protein targets and cuts the target genome under the guidance of gRNA, and the cut genome is repaired under the non-homologous recombination repair mechanism. Here Mutations can be introduced during the process to screen for rats with mutations in the target gene. The rat embryonic cells obtained in the present invention are used for in vitro culture or directly implanted into mother mice, express guide RNA and CAS protein and cut target genome, thereby screening rats with target gene mutation. Compared with the traditional method, the method of the present invention omits the process of screening ES cells in vitro, and the whole operation process only needs to directly introduce nucleic acid into embryos, and does not need to introduce ES cells into embryos, avoiding the possibility of ES cells being cultured in vitro. Irreversible differentiation that results when conditions are not met. Compared with ZFN and TALEN, the present invention eliminates the process of constructing and optimally screening the combination of ZFN and TALEN tandem base recognition modules, and the present invention allows targeting sequences to have a higher frequency of occurrence relative to ZFN or TALEN, thereby providing Precise genome editing offers more options. The gene mutant rats further obtained by using the rat embryo cells obtained in the present invention can be used for basic research in the field of life sciences, preclinical research for new drug development, and the like.

Wherein, the rat with the mutation of the target gene refers to crossing the rat with the mutation of the target gene with the wild-type rat, and then selecting the littermate heterozygous rat obtained from the crossing for selfing, and then from the above-mentioned obtained newborn large rat. Homozygous rats for mutations in the target gene were screened in mice.

The present invention also provides a rat embryo cell prepared according to the construction method of gene site-directed mutation of the present invention.

The present invention also provides a homozygous rat, which is a rat embryo cell prepared by the construction method of the gene site-directed mutation of the present invention. That is, the rat embryonic cells prepared according to the method of the present invention are used for in vitro culture or directly implanted into mother mice, express TALEN and cut the target genome, and screen for rats with mutations in the target gene; then hybridize them with wild-type rats , and then select the littermate heterozygous rats obtained from the above hybridization for selfing, and then screen the homozygous rats for the target gene mutation from the above obtained newborn rats.

The present invention uses RNA-guided endonucleases (RNA-guided endonucleases, RGENs) to achieve specific cutting of target gene sequences. RGENs are composed of chimeric guide RNA and Cas9 protein, in which the former is the fusion of CRISPR RNAs (crRNAs) and trans-activating crRNA (tracrRNA) in the naturally occurring type II CRISPR-Cas system into a single-stranded guide RNA (gRNA). ), thereby binding to the Cas9 protein and guiding the latter to specifically cut the target DNA sequence. The cleavage will form a double strand break (DSB), and after the damage is repaired by error-prone non-homologous end joining (NHEJ), there will be a 2/3 probability that the target gene will be damaged. Frameshift, so as to realize the knockout of the target gene. In the present invention, for example, implementing the gene knockout method in rat embryos includes the following steps:

(1) Find a suitable target sequence in the N-terminal coding sequence of the target rat gene.

(2) Obtain template DNA required for in vitro transcription of gRNA by overlapping primer PCR according to the above target sequence.

(3) The above template DNA was transcribed into RNA in vitro and purified.

(4) Simultaneously linearize the plasmid encoding the Cas9 protein and perform in vitro transcription.

(5) The above two RNAs were mixed and microinjected into rat zygotes formed by artificial insemination.

Further, after the above-mentioned steps of the present invention, the above-mentioned fertilized eggs were implanted into the fallopian tubes of pseudopregnant mother mice; the genomic DNA of the offspring rats obtained in the above-mentioned steps was extracted for genotype identification; the rats with frameshift mutations in the target gene were respectively Crossbreed with wild-type rats; self-breed the heterozygous rats obtained from the above-mentioned cross; identify the genotype of the above-mentioned self-crossed rats, and screen the homozygous rats for the frameshift mutation of the target gene.

The advantages of the present invention include: in the gene knockout technology mediated by ZFN or TALEN, it is necessary to construct expression plasmids with a length of several hundred or thousands of bases by repeated enzyme digestion and ligation, compared with the gRNA vector construction process of the present invention It is extremely simple, and two 20-bp complementary oligonucleotides containing the target can be directly synthesized and ligated into the gRNA expression plasmid after annealing. In the selection of available target sequences, ZFNs generally need to optimize screening from multiple candidate targets. The target selection of TALEN is also subject to the left and right targets. Base T restriction. However, the limitation of CRISPR target selection only comes from the fact that the three bases immediately adjacent to the 3′ end of the target must be in the form of 5′-NGG-3′, thus forming a PAM (protospacer adjacent motif) structure recognized by Cas9 itself. This opens up the possibility of more precise and flexible editing of the genome. After optimizing the concentration of RNA used for microinjection, the mutation rate of F0 generation rats obtained by the present invention can reach 78%, which is significantly higher than that achieved by using ZFN or TALEN. By injecting more than one gRNA at a time, the present invention can simultaneously edit multiple genes in fertilized rat eggs at one time. Through a single injection of gRNAs targeting two adjacent targets, the present invention can achieve the deletion of larger fragments of genomic DNA. Compared with the traditional method of knocking out or changing the target gene through homologous recombination in rat embryonic stem cells, the present invention avoids the long and difficult process of constructing the targeting vector. Secondly, in the present invention, the progeny rats obtained by microinjection of in vitro transcribed guide RNA nucleic acid sequence and CAS protein coding nucleic acid sequence, as described in the examples, have a probability of base deletion of one allele as high as 86%. However, the prior art and literature show that through homologous recombination and subsequent screening in rat embryonic stem cells, the probability of site-specific knockout does not exceed 15-65%. The selected ES cells need to be microinjected into embryos to produce chimeric rats. Only when chimeric rats undergo germline transmission can they obtain heterozygous rats for specific gene knockout. However, due to the irreversible differentiation of ES cells due to changes in culture conditions during the selection process, it is likely that the progeny cells differentiated from mutant ES cells cannot enter the germline and cannot obtain knockout rats. In the present invention, there is no problem of ES cell differentiation by injecting embryos at the single-cell stage, so through repeated experiments, it is generally possible to obtain a gene-knockout rat strain. The high mutation efficiency realized by the present invention has great significance in saving experiment time and input of manpower and material resources. Moreover, this high mutation efficiency will also facilitate the application of this technology in large animals that lay fewer eggs, have longer growth cycles, and have high economic value per animal. Again, the present invention directly microinjects the in vitro transcribed guide RNA nucleic acid sequence and CAS protein mRNA into artificially fertilized rat zygotes, avoiding the process of long cycle, heavy labor, and screening of rat embryonic stem cells with homologous recombination. . In addition, in the present invention, it only takes less than one week to construct the gRNA plasmid and carry out in vitro transcription, and it only takes about one month from microinjection to identification of heterozygous rats with frameshift in the target gene, while the traditional method The cycle for homologous recombination in rat embryonic stem cells to construct knockout rats usually takes at least a year. The method of the invention greatly shortens the experiment cycle and is conducive to the efficient advancement of research.

The present invention rapidly constructs a gene knockout rat method by targeting any genomic DNA in the rat embryo and making it change, and directly introduces the in vitro transcribed microinjection that recognizes and cuts the target gene into fertilized rat eggs The guide RNA nucleic acid sequence and the CAS protein-encoding nucleic acid sequence cause a frameshift of the target gene through DNA double strand break (double strand break, DSB) and the resulting non-homologous end joining (NHEJ) repair , so as to realize rapid and efficient gene site-directed mutation in rat embryonic cells. By using the present invention, F0 generation heterozygous or homozygous rats with target gene knockout can be further obtained. The invention overcomes the problem that artificial Zinc Finger Nucleases (Zinc Finger Nucleases, ZFN) cannot recognize any target DNA sequence by injecting fertilized eggs of rats, and the recognition characteristics are often affected by upstream and downstream sequences, and the resulting ZFN construction complex and expensive optimization screening process in the process. It overcomes the cumbersome splicing process in the assembly process of TALEN modules and the limited flexibility of TALEN target selection. Compared with the traditional method of knocking out target genes by using homologous recombination targeting vectors in rat embryonic stem cells, the present invention does not need to construct homologous recombination targeting vectors, does not need to screen ES targeting cells, has a high proportion of mutants, and is easy to operate , the experimental cost and cycle are greatly reduced and other advantages.

Description of drawings

Figure 1 is a schematic diagram of the working principle of the CRISPR/Cas system.

Figure 2 is a schematic diagram of Cas9mRNA and guide RNA expression vectors.

Fig. 3 is the T7E1 enzyme digestion electrophoresis diagram of the corresponding PCR product of neonatal rats obtained by microinjection of Cas9mRNA/MC4R-gRNA rats.

Figure 4 is a schematic diagram of base deletions in neonatal rats obtained by microinjection of Cas9mRNA/MC4R-gRNA.

Figure 5 is a schematic diagram of the base deletion situation of the F1 generation rats born to No. 12 first-built rats.

Fig. 6 is a schematic diagram of the prokaryotic expression vector of CAS nuclease.

Fig. 7 is a schematic diagram of base deletion in neonatal rats obtained by microinjection of Cas9 protein/MC4R-gRNA and Cas9 protein/MC3R-gRNA.

Detailed ways

The present invention will be described in further detail in conjunction with the following specific examples and accompanying drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present utility model, and the appended claims are the protection scope. The process, conditions, reagents, experimental methods, etc. for implementing the present invention are general knowledge and common knowledge in the art except for the content specifically mentioned below, and the present invention has no special limitation content. As described by Sambrook et al., Molecular Cloning, A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

The method for constructing gene site-directed mutation in rat embryonic cells of the present invention is a method for targeting any genomic DNA and making it change. The method includes: introducing into rat cells the encoding nucleic acid of guideRNA that recognizes the target gene And biologically active substances such as CRISPR/Cas9 protein and encoding nucleic acid, protein or artificially modified virus, so as to recognize and cut the target genomic DNA sequence. Then, the cells are cultured in vitro or directly transferred into the oviduct or uterus of a suitable female mouse, so that the transcriptional activator-like nuclease is expressed and the target genomic DNA near the cleavage site is double-broken, and then the DNA Repair the broken site.

Wherein, the repair methods include: (a) non-homologous end joining repair. Non-homologous end joining repair results in genetic mutations (base insertions, deletions) being introduced into the target genomic sequence. (b) Homologous recombination repair. Homologous recombination repair allows the introduction of donor exogenous DNA sequences into the target genomic DNA sequence, resulting in changes in the endogenous target gene sequence.

The construction method of gene site-directed mutation in rat embryonic cells of the present invention comprises the following steps:

(1) Construction of NLS-hCas9-NLS in vitro transcription vector

On the vector, introduce the SP6 promoter and Kozak sequence through the NcoI restriction endonuclease site overlapping with the start codon of the NLS-hCas9-NLS coding sequence; synthesize single-stranded oligonucleotides P1 and P2; use PNK phosphoric acid The enzyme adds a 5' phosphate group to the above single-stranded oligonucleotide, and anneals it to the vector that has been digested with NcoI and dephosphorylated; select the SP6-Cas9 plasmid with the correct insertion direction of the SP6 promoter;

Wherein, the P1 primer sequence: CATGGATTTAGGTGACACTATAGAAGAGGCCGCCAC;

P2 primer sequence: CATGGTGGCGGCCTCTTCTATAGTGTCACCTAAATC;

(2) Preparation of Cas9 nuclease mRNA

Select the SP6-Cas9 plasmid with the correct insertion direction, and linearize it with the NotI endonuclease downstream of the Cas9 coding region; use the SP6mMESSAGEmMACHINE Kit kit for in vitro transcription;

(3) Determine the Cas9 target site

Identify "GGCTGCTGCGGTTCCAGAGG" as the target sequence;

(4) Preparation of guide RNA in vitro transcription template

Synthesize single-stranded oligonucleotides P3, P4, P5, and P6 for primer overlap PCR;

in,

P3 primer sequence: GATCACTAATACGACTCACTATAGGCTGCTGCGGTTCCAGAGGGTTTTAGAGCTAGAAAT;

P4 primer sequence: AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC;

P5 primer sequence: GATCACTAATACGACTCAC;

P6 primer sequence: AAAAAAGCACCGACTCGGTGCC;

(5) Using the above-mentioned PCR product as a template, perform in vitro transcription, and mix it with the prepared Cas9 nuclease mRNA;

Mix the guide RNA and Cas9 mRNA obtained by in vitro transcription, dilute with TE, so that the final concentration of guide RNA is 25 ng/μl, and the final concentration of Cas9 mRNA is 50 ng/μl, and inject it into the prokaryotic part of fertilized rat eggs in vitro, and culture in vitro for 1-2 hours .

The method for constructing gene location mutation in rat embryonic cells of the present invention comprises gene mutation or gene knockout, and its steps include:

(1) Find a suitable target sequence in the N-terminal coding sequence of the target rat gene.

(2) Synthesize two complementary single-stranded oligonucleotides according to the above-mentioned target sequence, and anneal into the gRNA expression vector.

(3) The above expression vector was linearized, transcribed into RNA in vitro, and purified.

(4) Simultaneously linearize the plasmid encoding the Cas9 protein and perform in vitro transcription.

(5) The above two RNAs were mixed and microinjected into rat zygotes formed by artificial insemination.

Further, the fertilized eggs were implanted into the fallopian tubes of pseudopregnant mother mice; the genomic DNA of offspring rats obtained in the above steps was extracted for genotype identification; the rats with frameshift mutations in the target gene were crossed with wild-type rats; The littermate heterozygous rats obtained from the above hybridization are selfed; the genotypes of the rats obtained from the self-crossing are identified, and homozygous rats with a frameshift mutation of the target gene are screened.

In the present invention, rat cells are rat embryonic cells. Rat embryos include rat unicellular fertilized eggs or multicellular rat embryos. The rat target genome sequence is the genome sequence of the coding gene, low repetitive sequence, moderate repetitive sequence, high repetitive sequence, low copy sequence, transcription active sequence or non-transcriptional sequence among genes.

Example 1 Construction of Mc4r Gene Knockout Rats by Injecting RNA in Rat Cells

1. Construction of NLS-hCas9-NLS in vitro transcription vector

As shown in Figure 2, the Cas9 protein expression module consists of the SP6 promoter sequence from the 5' end to the 3' end, the N-terminal nuclear localization signal (Nuclear localization sequence, NLS), the humanized Cas9 coding DNA sequence, and the C-terminal core. The positioning signal is composed of polyadenylic acid (polyA). The specific construction strategy is, on the basis of the vector pX260 (Addgene plasmid #42229), introduce the SP6 promoter and the Kozak sequence through the NcoI restriction endonuclease site overlapping with the start codon of the NLS-hCas9-NLS coding sequence. That is, synthesize single-stranded oligonucleotides P1 (SEQ ID NO.1) and P2 (SEQ ID NO.2), use PNK phosphatase to add 5' phosphate groups to the above-mentioned single-stranded oligonucleotides, and anneal them Ligated with the pX260 vector that was digested with NcoI and dephosphorylated. Several clones were picked and sequenced to select the plasmid with the correct insertion direction of the SP6 promoter.

P1 primer sequence: CATGGATTTAGGTGACACTATAGAAGAGGCCGCCAC (SEQ ID NO.1)

P2 primer sequence: CATGGTGGCGGCCTCTTCTATAGTGTCACCTAAATC (SEQ ID NO.2)

2. Preparation of Cas9 nuclease mRNA

Select the SP6-Cas9 plasmid with the correct insertion direction, and use the NotI endonuclease downstream of the Cas9 coding region for linearization. The linearized SP6-Cas9 fragment was recovered by phenol-chloroform extraction, and the SP6mMESSAGEmMACHINE Kit kit was used for in vitro transcription. Precipitate the mRNA obtained by in vitro transcription with the LiCl solution provided in the kit, and resuspend with TE for microinjection.

3. Prediction of Cas9 target sites

Obtain the target DNA sequence from NCBI, in this embodiment, it is the CDS region of the first exon of rat Mc4r, copy and paste the target sequence into a Word document, use the "find" function (Ctrl+F), and search for the content Enter "GG???????????????????????GG" in the text box (the first 20 bases are used for complementary pairing with the non-coding strand of the target DNA) to search for non-coding DNA Target-eligible sequences in the template strand. The above base composition is because the product transcribed by the T7 promoter must start with "GG" to be efficiently transcribed. At the same time, the type II CRISPR system requires that the proto-spacer-adjacent motifs (PAM) immediately adjacent to the 3' end of the target conform to the composition of "NGG". According to the same principle, you can also search for "CC???????????????????????CC" in the reverse complementary sequence of the candidate target region to find potential targets. Finally, "GGCTGCTGCGGTTCCAGAGG" was selected as the target sequence.

4. Preparation of guide RNA in vitro transcription template

As shown in Figure 2, the guide RNA in vitro transcription template consists of a T7 promoter sequence, a target-specific sequence, and a guide RNA backbone part from the 5' to the 3' end. After determining the target, synthesize single-stranded oligonucleotides P3 (SEQ ID NO.3), P4 (SEQ ID NO.4), wherein the single-stranded oligonucleotide P3 contains T7 promoter from 5' to 3' end Subsequence, target-specific sequence, and bases at the 5' end of the guide RNA backbone. The single-stranded oligonucleotide P4 encodes the guide RNA backbone part, and its 3' end is complementary to the 3' end of the single-stranded oligonucleotide P3. Synthetic single-stranded oligonucleotides P5 and P6 have the same base sequence as the 5' end of single-stranded oligonucleotides P3 and P4 respectively. Primer overlap PCR was performed using single-stranded oligonucleotides P3, P4, P5, P6. The PCR product was purified by phenol-chloroform extraction and isopropanol precipitation, resuspended with RNase Free ddH2O, and the concentration was measured.

P3 primer sequence (SEQ ID NO.3): GATCACTAATACGACTCACTATAGGCTGCTGCGGTTCCAGAGGGTTTTAGAGCTAGAAAT

P4 primer sequence (SEQ ID NO.4): AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC

P5 primer sequence: GATCACTAATACGACTCAC

P6 primer sequence: AAAAAAGCACCGACTCGGTGCC

5. Preparation of Cas9 nuclease mRNA

Using the above PCR product as a template, perform in vitro transcription according to the instructions of the in vitro Transcription T7Kit (Takara #6140) kit. Transcripts were purified by phenol-chloroform extraction and isopropanol precipitation, and resuspended using TE for microinjection.

6. Microinjection of guide RNA and Cas9 mRNA

The guide RNA and Cas9 mRNA obtained by in vitro transcription were mixed and diluted with TE for microinjection, so that the final concentration of guide RNA was 25 ng/μl, and the final concentration of Cas9 mRNA was 50 ng/μl. The above-mentioned mRNA solution is injected into the prokaryotic part of the in vitro fertilized rat zygote using a microinjection instrument to construct a specific rat embryonic cell (zygote), which is to complete the construction of the site-directed mutation of the gene in the rat embryonic cell of the present invention.

The following is used to identify whether the method for constructing gene site-directed mutation in rat embryonic cells of the present invention is successfully implemented.

(1) Genotype identification of neonatal rats

After the rat embryonic cells (fertilized eggs) obtained in step 6 were cultured in vitro for 1-2 hours, the surviving fertilized eggs were transplanted into the oviducts of pseudopregnant mother mice. One week after birth, the toes of the newborn rats were clipped and the genomic DNA was extracted. PCR primer P7 (SEQ ID NO.7) was designed upstream of the target site, and PCR primer P8 (SEQ ID NO.8) was designed downstream, and the rat genomic DNA to be identified was used as a template for PCR amplification. After the PCR reaction, the product was analyzed. Purification, denaturation, slow annealing, adding T7Endonuclease I that can recognize and cut at non-matching sites, and perform agarose gel electrophoresis. Fig. 3 is an electrophoresis diagram of PCR product T7E1 corresponding to neonatal rats obtained by microinjection. As shown in Figure 3, a total of 13 out of 15 newborn rats had mutations at the target site. in,

P7 primer sequence: TACTGTTTAGCAGGGTGATGACG (SEQ ID NO.7)

P8 primer sequence: GAAGAGACCAACAACTCCTTTGC (SEQ ID NO.8)

The PCR products of the T7E1-positive rats were connected into the pMD18T vector, and several clones were picked to extract the plasmids and sequenced. Figure 4 is the result of the comparison of base deletions in first-built rats. As shown in Figure 4, the underlined part is the protospacer (protospacer), that is, the region where the target is located, and the following TGG is the proto-spacer-adjacent motifs (PAM). Each dash represents a missing base.

(2) The first establishment of germline inheritance of Mc4r gene mutation in rats

The No. 12 Mc4r gene mutant rat was selected and crossed with the wild-type rat, and the genotype of the newborn rat was identified by the above method. Figure 5 is a schematic diagram of the base deletion situation of the F1 generation rats born to No. 12 first-built rats. As shown in Figure 5, 3 mice out of 6 newborn F1 rats carried the mutation. Among them, the underlined part is the protospacer (protospacer), that is, the region where the target is located, and TGG behind it is the proto-spacer-adjacent motifs (PAM). Each dash represents a missing base. It is proved that Mc4r gene mutation can be inherited stably.

Example 2 Construction of Mc3r/Mc4r knockout rats by injecting CAS protein and gRNA in rat cells

1. Construction of CAS nuclease prokaryotic expression vector PET-28a-H6-3FLAG-NLS-CAS9-NLS

As shown in Figure 6, the prokaryotic expression vector of Cas9 fusion protein consists of T7 promoter sequence, His6Tag, 3×Flag, N-terminal nuclear localization signal (Nuclear localization sequence, NLS1), humanized The Cas9-encoded DNA sequence consists of the C-terminal nuclear localization signal NLS2. First, based on the NLS-hCas9-NLS in vitro transcription vector, His6 and 3×FLAG tags, as well as a new N-terminal NcoI restriction enzyme site and a C-terminal EcoRI restriction enzyme site were introduced by overlapping PCR. A new H6-3FLAG-NLS-CAS9-NLS junction was inserted through the NcoI and EcoRI restriction sites on PET-28a and used as translation initiation for protein expression through the start codon contained in the NcoI restriction site location.

P9 primer sequence: CCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGAC

P10 primer sequence: GATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGG

P11 primer sequence: GAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCCAGCAGCCGACAAGAAGTACAGCATCG

P12 primer sequence: GGCAAAAAAGAAAAAGTAAGAATTCCTA

2. Expression and purification of Cas9 nuclease

The constructed and identified PET-28a-H6-3FLAG-NLS-CAS9-NLS plasmid was transfected into Rosseta DE3 host competent cells. Activate the cells at 24°C until the OD value is 0.8-1.0, then add IPTG for induction, so that the final concentration of IPTG is 0.1mM, and induce for 17 hours at 16°C. After the cells were ultrasonically disrupted, the supernatant was collected and purified with a nickel column for later use.

3. Prediction of Cas9 target sites

As in Example 1, "GGCTGCTGCGGTTCCAGAGG" was finally selected as the targeting sequence of Mc4r, and "GGGCTGCAGGGTGCTGGGAG" was selected as the targeting sequence of Mc4r.

4. Preparation of guide RNA in vitro transcription template

With embodiment 1.

5. Microinjection of guide RNA and Cas9 protein

The guide RNA obtained by in vitro transcription and the prokaryotic expressed Cas9 protein were mixed and diluted with TE for microinjection, so that the final concentration of the guide RNA was 12.5 ng/μl, and the final concentration of the Cas9 protein was 5 ng/μl. The above-mentioned mRNA solution is injected into the prokaryotic part of the in vitro fertilized rat zygote using a microinjection instrument to construct a specific rat embryonic cell (zygote), which is to complete the construction of the site-directed mutation of the gene in the rat embryonic cell of the present invention.

The following is used to identify whether the method for constructing gene site-directed mutation in rat embryonic cells of the present invention is successfully implemented.

(1) Genotype identification of neonatal rats

With embodiment 1. After the rat embryonic cells (fertilized eggs) obtained in step 6 were cultured in vitro for 1-2 hours, the surviving fertilized eggs were transplanted into the oviducts of pseudopregnant mother mice. A total of 3 of the 5 newborn rats had mutations at the target site, and 2 of them had double gene mutations at the same time.

Wherein the PCR primers for Mc4r identification are: P7 primer sequence: TACTGTTTAGCAGGGTGATGACG

P8 primer sequence: GAAGAGACCAACAACTCCTTTGC

PCR primers for Mc3r identification are: P13 primer sequence AATCTAGACTGGACAGCATCCAC

P14 primer sequence TGATAACCACGATCATGATGGTC

The PCR products of the T7E1-positive rats were connected into the pMD18T vector, and several clones were picked to extract the plasmids and sequenced. Figure 7 is the result of the comparison of base deletions in first-built rats. As shown in Figure 7, the underlined part is the protospacer (protospacer), that is, the region where the target is located, and the following TGG is the proto-spacer-adjacent motifs (PAM). Each dash represents a missing base.

Claims (6)

1. A method for constructing gene site-directed mutation in rat embryonic cells, characterized in that it comprises: (1) Determine the target sequence targeted by the CRISPR-Cas system in the rat target genome sequence; (2) Design and construct a gRNA nucleic acid sequence that can recognize and guide the CAS9 recombinant protein to the target gene target sequence; (3) After the gRNA nucleic acid sequence is mixed with the CAS9 recombinant protein, it is introduced into the nucleus of the rat embryo by microinjection; wherein, In the step (1), search for the target sequence in the non-template strand according to "GGNNNNNNNNNNNNNNNNNNNNGG" in the candidate target region; or search for the target sequence by "CCNNNNNNNNNNNNNNNNNNNNCC" in the reverse complementary sequence of the candidate target region; In the step (2), the gRNA nucleic acid sequence can specifically bind to the CAS protein and target the target sequence; In the step (3), the gRNA nucleic acid sequence is pre-transcribed into RNA in vitro and purified. 2. The method according to claim 1, wherein said CAS9 recombinant protein is prepared by CAS nuclease prokaryotic expression vector PET-28a-H6-3FLAG-NLS-CAS9-NLS, wherein said H6-3FLAG-NLS-CAS9- NLS is composed of T7 promoter sequence, His6Tag, 3×Flag, N-terminal nuclear localization signal NLS1, humanized Cas9 coding DNA sequence, and C-terminal nuclear localization signal NLS2 from the 5' end to the 3' end. The NcoI and EcoRI restriction sites on -28a insert the H6-3FLAG-NLS-CAS9-NLS connection into it and use the start codon contained in the NcoI restriction site as the translation initiation site for protein expression, Thus, the prokaryotic expression vector PET-28a-H6-3FLAG-NLS-CAS9-NLS of the CAS nuclease is obtained. 3. The method of claim 1, wherein the gene for site-directed mutation is the Mc3r or Mc4r gene. 4. The method according to any one of claims 1-3, wherein in the step (3), the DNA break site formed by cutting the target genome is repaired by error-prone non-homologous end joining or Repaired by homologous recombination. 5. The method according to any one of claims 1-3, wherein the mutation of the target gene is base insertion, deletion, alteration, frameshift mutation or knockout. 6. The method according to any one of claims 1-3, wherein the rat embryo cells are rat single-cell fertilized eggs or multi-cell rat embryos.