CN106957857A - A kind of method that utilization CRISPR/Cas9 systems knock out goat MSTN and FGF5 gene jointly - Google Patents

Abstract

The invention provides a kind of method that utilization CRISPR/Cas9 systems knock out goat MSTN and FGF5 gene jointly.The present invention obtains two segment DNA sequences of the sgRNA cog regions for goat MSTN Second Exons and the 3rd extron first, and two segment DNA sequences of the sgRNA cog regions for goat FGF5 First Exons, separately design and synthesize corresponding sgRNA oligonucleotide sequences；Then the in-vitro transcription carrier for MSTN Second Exons and the 3rd extron, FGF5 First Exons and the promoter containing T7 is built respectively；Cas9 mRNA and sgRNA are obtained using Cas9 and sgRNA in-vitro transcription carrier, the transgenic goat knocked out jointly available for production MSTN and FGF5.

Description

A co-knockout goat MSTN and FGF5 gene using CRISPR/Cas9 system method

technical field

The invention relates to the field of animal genetic engineering and genetic modification, in particular to a method for jointly knocking out goat MSTN and FGF5 genes by using a CRISPR/Cas9 system.

Background technique

The CRISPR/Cas system is an acquired immune system in bacteria and archaea through RNA-mediated specific cutting of exogenous genetic material, which can be used to fight against invading viruses and exogenous DNA. The CRISPR/Cas9 system confers immunity by integrating fragments of invading phage and plasmid DNA into CRISPR and using corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences.

The type II CRISPR/Cas system, CRISPR/Cas9, has been shown to efficiently cut any given DNA in a test tube. Compared with traditional ZFN and TALEN technologies, CRISPR/Cas9 has higher efficiency, less restriction on sequence selection (only GG appears on the genome), and its construction process is simple. It only needs to construct a suitable sgRNA for each gene. However, CRISPR/Cas9 can cause serious off-target effects in mammalian cells. The use of Cas9 nickase plus two opposite PAMs, sgRNAs that are relatively close and can be combined on different strands can greatly reduce the off-target efficiency.

Traditional breeding methods have disadvantages such as long breeding period and limited number of selected traits at one time. Molecular marker-assisted selection is still a problem with rapid theoretical development but is difficult to apply to production practice. The combination of transgenic breeding technology and traditional breeding technology is particularly important. An important gene related to growth—MSTN, namely myostatin, also known as growth/differentiation factor-8 (GDF-8). In mammals, the MSTN gene is mainly expressed in skeletal muscle, and the mutation site (A>G) of the gene in the untranslated region produces an interference target site, which inhibits the translation process and regulatory function of the MSTN gene, thus affecting animal Muscle development. FGF5, namely fibroblast growth factor 5, has been proved to be an important factor affecting hair length, which hinders hair growth by inhibiting the activity of hair follicle dermal papilla cells. Through the CRISPR/Cas9 system, the MSTN gene that inhibits muscle growth and the FGF5 gene that inhibits cashmere growth in the goat genome are jointly knocked out, and the production of goats with strong muscles, long hair, and meat is of great significance to the long-term development of my country's animal husbandry. .

Myostatin (MSTN) is a conserved negative regulator of mammalian skeletal muscle mass. However, whether precise knockout of MSTN can be achieved in livestock and can be safely used to improve meat production has not yet been proven.

Contents of the invention

The invention provides a method for jointly knocking out goat MSTN and FGF5 genes by using the CRISPR/Cas9 system.

In order to achieve the above object, the technical scheme that the present invention takes is:

A method utilizing CRISPR/Cas9 system to knock out goat MSTN and FGF5 gene jointly, is characterized in that, comprises the following steps:

(1) Construct the in vitro transcription vector of sgRNA specifically targeting the second exon and the third exon of MSTN and the first exon of FGF5; obtain the second exon and the third exon of MSTN by in vitro transcription sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F of the first exon of FGF5;

(2) an in vitro transcription vector for transcribing the Cas9 protein in vitro to obtain Cas9 mRNA;

(3) Purify the sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9mRNA in step (1) and step (2) and measure the concentration, mix them, inject them into the cytoplasm of goat fertilized eggs, and then culture them in vitro Transplanted into oviducts of congenic female goats to produce transgenic goats that co-knock out MSTN and FGF5.

After mixing the above sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9mRNA, the final concentration is Cas9mRNA 20ng/μL, sgRNA-1M 5ng/μL, sgRNA-2M 5ng/μL, sgRNA-1F 5ng/μL μL, sgRNA-2F5ng/μL.

A kind of above-mentioned method utilizing CRISPR/Cas9 system to jointly knock out goat MSTN and FGF5 gene is characterized in that, the DNA sequence of described sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F in vitro The transcription vector is the in vitro transcription vector Puc57-T7-gRNA.

The method for knocking out goat MSTN and FGF5 genes by using the CRISPR/Cas9 system described above is characterized in that two pairs of sgRNA-1M specifically targeting the second exon and the third exon of MSTN , sgRNA-2M oligonucleotide sequence, two pairs of sgRNA-1F and sgRNA-2F oligonucleotides of the first exon of FGF5.

The present invention also provides the following applications of the method for jointly knocking out goat MSTN and FGF5 genes using the CRISPR/Cas9 system:

(1) Simultaneously transfect sgRNA-1M, sgRNA-2M or Cas9mRNA into cells to study the function of MSTN gene;

(2) Inject sgRNA-1M, sgRNA-2M together or alone with Cas9mRNA into fertilized eggs, and then use embryo transfer to produce transgenic goats targeted to knock out the MSTN gene;

(3) transfect sgRNA-1M, sgRNA-2M together or alone with Cas9mRNA into cells, and use positive cells after screening as nuclear donor cells to produce transgenic goats with targeted knockout of MSTN gene by nuclear transfer;

(4) Transfect sgRNA-1F and sgRNA-2F together or separately with Cas9mRNA into cells simultaneously for studying the function of FGF5 gene;

(5) Inject sgRNA-1F, sgRNA-2F together or alone with Cas9mRNA into fertilized eggs, and then use embryo transfer to produce transgenic goats targeted to knock out the FGF5 gene;

(6) Transfect sgRNA-1F, sgRNA-2F together or alone with Cas9mRNA into cells, and use positive cells after screening as nuclear donor cells to produce transgenic goats with targeted knockout of the FGF5 gene by nuclear transfer.

(7) Simultaneously inject sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9mRNA into fertilized eggs, and then use embryo transfer to produce transgenic goats that jointly knock out MSTN gene and FGF5 gene;

(8) Simultaneously transfect sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9mRNA into cells, and use positive cells as nuclear donor cells after screening to produce co-knockout of MSTN gene and FGF5 gene through embryo transfer transgenic goats;

(9) Simultaneously inject any two or more sgRNAs of sgRNA-1M, sgRNA-2M, sgRNA-1F, and sgRNA-2F with Cas9mRNA into fertilized eggs, and then use embryo transfer to produce specific targeted knockout transgenes goat;

(10) Transfect any two or more sgRNAs of sgRNA-1M, sgRNA-2M, sgRNA-1F, and sgRNA-2F with Cas9mRNA into cells at the same time, and use positive cells as nuclear donor cells after screening for embryo transfer. Production of transgenic goats with specific targeted knockouts.

The working principle of this system is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which guides the nuclease Cas9 protein at the sequence target site paired with crRNA Shears double-stranded DNA. By artificially designing these two RNAs, it is possible to transform and form a guiding sgRNA (singleguide RNA), which is sufficient to guide the site-specific cutting of DNA by Cas9. As an RNA-guided dsDNA-binding protein, the Cas9 effector nuclease is the first known unifying factor capable of co-localizing RNA, DNA, and protein, thus possessing enormous engineering potential. The protein is fused with nuclease-free Cas9 (Cas9nuclease-null), and the appropriate sgRNA is expressed to target any dsDNA sequence, and the end of the sgRNA can be connected to the target DNA without affecting the binding of Cas9. Therefore, Cas9 can bring any fusion protein and RNA at any dsDNA sequence, which brings great potential for the research and transformation of organisms.

Gene sequence of the present invention:

1. A method for jointly knocking out goat MSTN and FGF5 genes using the CRISPR/Cas9 system, aiming at the specific target site sequences of the second exon and the third exon of goat MSTN and the first exon of FGF5, characterized in that, Its location in the genome is shown in Table 1.

Table 1

2. Two pairs of sgRNA-1M and sgRNA-2M oligonucleotide sequences specifically targeting the second exon and the third exon of MSTN, and two pairs of sgRNA-1F and sgRNA-2F targeting the first exon of FGF5 The oligonucleotide sequence is characterized in that its targeting site is shown in Table 1, and its base sequence is shown in Table 2.

sgRNA-1M top strand TAGGTCTCAGATATATCCACAGT sgRNA-1M bottom strand AAACACTGTGGATATATCTGAGA sgRNA-2M top strand TAGGATTTTGAAGCTTTTGGAT sgRNA-2M bottom strand AAACATCCAAAAGCTTCAAAAT sgRNA-1F top strand TAGGCTGCCACGGAGAGGAACC sgRNA-1F bottom strand AAACGGTTCCCTCTCCGTGGCAG sgRNA-2F top stratnd TAGGCAGCCCTCTACTGCAGAGT sgRNA-2F bottom strand AAACACTCTGCAGTAGAGGCTG

Table 2

3. A method for jointly knocking out goat MSTN and FGF5 genes using the CRISPR/Cas9 system. Table 3 shows the primer sequences used for the detection and amplification of target fragments of MSTN and FGF5;

table 3

detailed description

In order to make the objects and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The embodiment of the present invention provides a method for jointly knocking out goat MSTN and FGF5 genes using the CRISPR/Cas9 system, comprising the following steps:

(1) Construct the in vitro transcription vector of sgRNA specifically targeting the second exon and the third exon of MSTN and the first exon of FGF5; obtain the second exon and the third exon of MSTN by in vitro transcription sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F of the first exon of FGF5;

(2) an in vitro transcription vector for transcribing the Cas9 protein in vitro to obtain Cas9 mRNA;

(3) Measure concentration after purifying sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9mRNA of step (1) and step (2), sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA -2F and Cas9 mRNA were mixed, injected into the cytoplasm of fertilized goat eggs, and then cultured in vitro and transplanted into the oviduct of the same female goat to produce transgenic goats that co-knock out MSTN and FGF5.

After the sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9mRNA are mixed, the final concentration is Cas9mRNA 20ng/μL, sgRNA-1M 5ng/μL, sgRNA-2M 5ng/μL, sgRNA-1F Sng/μL μL, sgRNA-2F5ng/μL.

The in vitro transcription vector of the DNA sequences of sgRNA-1M, sgRNA-2M, sgRNA-1F and sgRNA-2F is Puc57-T7-gRNA.

Two pairs of sgRNA-1M and sgRNA-2M oligonucleotide sequences specifically targeting the second exon and third exon of MSTN, two pairs of sgRNA-1F and sgRNA-2F oligonucleotides targeting the first exon of FGF5 Nucleotides.

Step 1: Construction of CRISPR/Cas9 system for MSTN gene and FGF5 gene

1. According to the goat genome sequence in NCBI, select the second exon and the third exon of the MSTN gene in the goat genome as the target site to design sgRNA-1M, sgRNA-2M, and the first exon of the FGF5 gene as the target site Design sgRNA-1F and sgRNA-2F, the target site sequence is shown in Table 1, and the sgRNA sequence is shown in Table 2.

Table 1

Table 2

2. Construction of pUC57-T7-gRNA vector containing specific sgRNA sequence: (1) Design and synthesis of sgRNA-1M and sgRNA-2M that recognize the second exon and third exon of MSTN gene and the first one that recognizes FGF5 gene One exon was used as the target site to design sgRNA-1F and sgRNA-2F; (2) The synthesized sgRNA-1M, sgRNA-2M, sgRNA-1F, and sgRNA-2F paired oligonucleotides were annealed in vitro; ( 3) Digest and ligate sgRNA-1M, sgRNA-2M, sgRNA-1F, and sgRNA-2F through the BsaI site, insert them into pUC57-T7-gRNA, and name them pUC57-T7-sgRNA-1M, pUC57- T7-sgRNA-2M, pUC57-T7-sgRNA-1F, pUC57-T7-sgRNA-2F;

The CRISPR/Cas9 system for MSTN gene and FGF5 gene is: in vitro transcription vector pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1F, pUC57-T7-sgRNA-2F and pST1374 -NLS-flag-linker-Cas9.

Step 2: In vitro transcription

In vitro transcription vector pST1374-NLS-flag-linker- Cas9 performs in vitro transcription mediated by the T7 promoter. (1) Linearize pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1F, pUC57-T7-sgRNA-2F respectively with Dra I, pST1374-NLS-flag-linker- Cas9 was linearized with Age1; (2) pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1F, pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-1F, pUC57-T7-sgRNA- 2F. In vitro transcription of pST1374-NLS-flag-linker-Cas9; (3) Purification of sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F, and Cas9 mRNA using the MEGAClear kit (Ambion) kit according to the instructions.

Step 3: Using CRISPR/Cas9 system mRNA targeting MSTN gene and FGF5 gene to produce gene-targeted goats that co-knock out MSTN gene and FGF5 gene

1. Pronuclear injection and embryo transfer

Embryos at the single-cell stage (about 14 hours after fertilization) were surgically collected from donor female goats that were mated naturally after estrus synchronization, and the premixed sgRNA-1M, sgRNA-2M, sgRNA- 1F, sgRNA-2F, Cas9mRNA mixture (final concentration of Cas9mRNA 20ng/μL after mixing, sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F 5ng/μL) was injected into the cytoplasm of goat fertilized eggs. After the injection, the fertilized eggs were transferred to Quinn's Advantage Cleavage Medium (Sage Biopharma, NJ, USA) and cultured at 37°C for 24 hours in vitro, and then transplanted to the junction of the ampulla and isthmus of the oviduct of recipient ewes to produce co-knockout of MSTN gene and FGF5 gene gene targeting goats.

2. Identification of gene-targeted goats with co-knockout of MSTN gene and FGF5 gene

After the recipient ewe gave birth, the lamb's blood sample was collected after the lamb was 1 week old, and the lamb's blood genomic DNA was extracted. Using the lamb blood genome as a template, primers for the second exon and the third exon of the MSTN gene and primers for the first exon of the FGF5 gene were designed respectively. The sequence is shown in Table 3. Amplification was carried out. The obtained PCR product was detected by agarose gel electrophoresis and the product system was recovered. The recovered PCR product was digested with T7EN1 enzyme. If there are more or less bands, the possibility of gene targeting is successful; the recovered PCR products are sent for sequencing and sequence analysis, combined with T7EN1 digestion results and sequencing results to determine positive individuals; the PCR products of positive individuals are cloned into T vectors, transformed Afterwards, the positive clones were picked and sequenced again, and the positive individuals with successful gene knockout and the change mode of bases in the positive individuals were further determined according to the sequencing results.

table 3

Although the present invention has been described in detail with general descriptions and specific embodiments above, they are only preferred embodiments of the present invention. It should be pointed out that those skilled in the art can make some improvements and modifications without departing from the principle of the invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (4)

1. A method utilizing CRISPR/Cas9 system to knock out goat MSTN and FGF5 gene jointly, is characterized in that, comprises the following steps: (1) Construct the in vitro transcription vector of sgRNA specifically targeting the second exon and the third exon of MSTN and the first exon of FGF5; obtain the second exon and the third exon of MSTN by in vitro transcription sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F of the first exon of FGF5; (2) an in vitro transcription vector for transcribing the Cas9 protein in vitro to obtain Cas9 mRNA; (3) Measure concentration after purifying sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9 mRNA of step (1) and step (2), sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and Cas9 mRNA were mixed, injected into the cytoplasm of fertilized goat eggs, and then cultured in vitro and transplanted into the oviducts of female goats of the same species to produce transgenic goats that co-knock out MSTN and FGF5. 2. a kind of method utilizing CRISPR/Cas9 system to knock out goat MSTN and FGF5 gene according to claim 1, is characterized in that, described sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F and After Cas9 mRNA is mixed, the final concentration is Cas9 mRNA 20ng/μL, sgRNA-1M 5ng/μL, sgRNA-2M 5ng/μL, sgRNA-1F 5ng/μL, sgRNA-2F 5ng/μL. 3. a kind of method utilizing CRISPR/Cas9 system to knock out goat MSTN and FGF5 gene according to claim 1, is characterized in that, described sgRNA-1M, sgRNA-2M, sgRNA-1F, sgRNA-2F The in vitro transcription vector of DNA sequence is pUC57-T7-gRNA. 4. a kind of method utilizing CRISPR/Cas9 system to knock out goat MSTN and FGF5 gene according to claim 1, it is characterized in that, specific targeting is two exons of MSTN second exon and the third exon. For sgRNA-1M, sgRNA-2M oligonucleotide sequences, two pairs of sgRNA-1F, sgRNA-2F oligonucleotides for the first exon of FGF5.