CN106957858A - A kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly - Google Patents

Abstract

The invention provides a kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly.The present invention obtain first the sgRNA cog regions for sheep MSTN Second Exons and the 3rd extron two segment DNA sequences, for the extrons of sheep ASIP the 5th sgRNA cog regions two segment DNA sequences, for sheep BCO2 Second Exons sgRNA cog regions two segment DNA sequences, separately design and with into corresponding sgRNA oligonucleotide sequences；Then build respectively for sheep MSTN Second Exons and the 3rd extron, for the extrons of sheep ASIP the 5th, for sheep BCO2 Second Exons, and the saRNA in-vitro transcription carriers of the promoter containing T7；Cas9 mRNA and sgRNA are obtained by in-vitro transcription using Cas9 and sgRNA in-vitro transcription carrier, the Transgenic Sheep that can be used for production MSTN, ASIP, BCO2 to knock out jointly is injected by embryonated egg.

Description

A co-knockout sheep MSTN, ASIP, BCO2 gene using CRISPR/Cas9 system method of cause

technical field

The invention relates to the field of animal genetic engineering and genetic modification, in particular to a method for jointly knocking out sheep MSTN, ASIP and BCO2 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 excision of exogenous genetic material. 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. Using 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.

ASIP, the agouti signaling protein gene, has been proved to play an important role in the melanin synthesis signaling pathway, and the high expression of ASIP gene is significantly correlated with the white coat of sheep. The ASIP protein expressed in the skin by ASIP competes with α-MSH (melanocyte-stimulating hormone) to bind to MC1R (melanocorticoid receptor 1), changing the structure of MC1R, inhibiting the cyclic adenosinase system and causing a decrease in cAMP levels, through The cascade of reactions inhibits the formation of eumelanin, producing pheomelanin. Overexpression of the ASIP gene may also reduce the synthesis of melanin and make the coat color appear white. The agricultural value of sheep is not only to provide mutton, wool and sheepskin are also important products of sheep, but the irregular color of sheep's coat seriously affects the quality of fur and the later dyeing process.

Through the CRISPR/Cas9 system, the MSTN gene that inhibits muscle growth, the ASIP gene that affects fur quality, and the BCO2 gene that causes yellow fat disease in the sheep genome are jointly knocked out through CRISPR/Cas9 to produce muscular, high-quality fur and vitality. Good sheep are of great significance to the long-term development of my country's animal husbandry.

Contents of the invention

The invention provides a method for jointly knocking out sheep MSTN, ASIP and BCO2 genes by using the CRISPR/Cas9 system.

In order to achieve the above object, the technical solution adopted by the present invention is: a method for jointly knocking out sheep MSTN, ASIP, and BCO2 genes using the CRISPR/Cas9 system, which is characterized in that it comprises the following steps:

(1) Construct the in vitro transcription vector of sgRNA specifically targeting the second exon and the third exon of MSTN, the fifth exon of ASIP, and the second exon of BCO2; obtain the second exon of MSTN through in vitro transcription sgRNA-1M and sgRNA-2M for the exon and the third exon, sgRNA-1A and sgRNA-2A for the fifth exon of ASIP, and sgRNA-1B and sgRNA-2B for the second exon of BCO2 in sheep;

(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-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B and Cas9mRNA in step (1) and step (2), measure the concentration, mix them, and inject them into sheep for fertilization In the egg cytoplasm, and then transplanted into the oviduct of female sheep of the same species after in vitro culture, it is used to produce transgenic sheep that co-knock out MSTN, ASIP, and BCO2 genes.

A method of using CRISPR/Cas9 system to knock out sheep MSTN, ASIP, and BCO2 genes according to the present invention is characterized in that sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA -2B mixed with Cas9 mRNA, the final concentration is Cas9 mRNA 20ng/μL, sgRNA-1M 5ng/μL, sgRNA-2M 5ng/μL, sgRNA-1A 5ng/μL, sgRNA-2A 5ng/μL, sgRNA-1B 5ng/μL , sgRNA-2B 5ng/μL.

A method of using CRISPR/Cas9 system to knock out sheep MSTN, ASIP, and BCO2 genes of the present invention is characterized in that the sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B The expression vector of sgRNA-2B is pUC57-T7-gRNA, and the in vitro transcription vector of Cas9 protein is pST1374-NLS-flag-linker-Cas9.

A method of using CRISPR/Cas9 system to knock out sheep MSTN, ASIP, and BCO2 genes according to the present invention is characterized in that the specific targeting is the second exon and the third exon of sheep MSTN sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A targeting the fifth exon of sheep ASIP, sgRNA-1B, sgRNA-2B targeting the second exon of sheep BCO2.

The present invention also provides the application of the method for knocking out sheep MSTN, ASIP and BCO2 genes by 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 sheep 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 sheep targeted to knock out the MSTN gene by nuclear transfer;

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

(5) Inject sgRNA-1A, sgRNA-2A together or alone with Cas9mRNA into fertilized eggs, and then use embryo transfer to produce transgenic sheep targeting knockout of the ASIP gene;

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

(7) Transfect sgRNA-1B and sgRNA-2B together or separately with Cas9mRNA into cells simultaneously for studying the function of BCO2 gene;

(8) Inject sgRNA-1B, sgRNA-2B together or alone with Cas9mRNA into fertilized eggs, and then use embryo transfer to produce transgenic sheep targeting knockout of the BCO2 gene;

(9) transfect sgRNA-1B, sgRNA-2B together or alone with Cas9mRNA into cells, and use positive cells after screening as nuclear donor cells to produce transgenic sheep with targeted knockout of BCO2 gene by nuclear transfer;

(10) Simultaneously inject sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B and Cas9mRNA into fertilized eggs, and then use embryo transfer to produce co-knockout MSTN, ASIP, BCO2 genetically modified sheep;

(11) Simultaneously transfect sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B and Cas9mRNA into cells, and use positive cells as nuclear donor cells after screening for production by embryo transfer Transgenic sheep with co-knockout of MSTN, ASIP and BCO2 genes;

(12) Inject any two or more sgRNAs of sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, and sgRNA-2B into fertilized eggs simultaneously with Cas9mRNA, and then use embryo transfer for Production of transgenic sheep with specific targeted knockout;

(13) Transfect any two or more sgRNAs of sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, and sgRNA-2B with Cas9mRNA into cells at the same time, and use positive cells after screening as Donor cells are used to produce specifically targeted knockout transgenic sheep by embryo transfer.

The working principle of the CRISPR/Cas9 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 to sequence targets paired with crRNA. Site cuts double-stranded DNA. By artificially designing these two RNAs, sgRNA (singleguide RNA) with a guiding effect can be engineered, which is sufficient to guide Cas9 to cut DNA at a specific site. 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 an appropriate sgRNA is expressed, which can 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 sheep MSTN, ASIP, and BCO2 genes by using the CRISPR/Cas9 system. The specific target site sequences of the MSTN, ASIP, and BCO2 genes are shown in Table 1.

Table 1

2. A method for jointly knocking out sheep MSTN, ASIP, and BCO2 genes using the CRISPR/Cas9 system. The sgRNA sequences designed for the MSTN, ASIP, and BCO2 genes are shown in Table 2.

MSTN sgRNA-1 top strand tagGTCTCAGATATATCCACAGT MSTN sgRNA-1bottom strand aaacACTGTGGATATATCTGAGA MSTN sgRNA-2 top strand TAGGATTTTGAAGCTTTTGGAT MSTN sgRNA-2 bottom strand aaacATCCAAAAGCTTCAAAAT ASIP sgRNA-1 top strand ccggCTTCAGGTTCCTTTCATCTC ASIP sgRNA-1bottom strand aaacGAGATGAAAGGAACCTGAAG ASIP sgRNA-2 top strand CCGGCAATTCTTCCATGAACCTGT ASIP sgRNA-2bottom strand aaacACAGGTTCATGGAAGAATTG BCO2 sgRNA-1 top strand ccgGTTAGAAGCGGTGCAATGCA BCO2 sgRNA-1bottom strand aaacTGCATTGCACCGCTTTCTAA BCO2sgRNA-2top strand ccGGTCGTCTCACGCTCGAGTCC BCO2sgRNA-2bottom strand aaacGGACTCGAGCTGAGACGA

Table 2

3. A method for jointly knocking out sheep MSTN, ASIP, and BCO2 genes using the CRISPR/Cas9 system. The primer sequences used for the detection of lambs produced by sheep with knockout MSTN, ASIP, and BCO2 genes are shown in Table 3.

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 sheep MSTN, ASIP, and BCO2 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, the fifth exon of ASIP, and the second exon of BCO2; obtain the second exon of MSTN through in vitro transcription sgRNA-1M and sgRNA-2M for the exon and the third exon, sgRNA-1A and sgRNA-2A for the fifth exon of ASIP, and sgRNA-1B and sgRNA-2B for the second exon of BCO2 in sheep;

(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-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B and Cas9mRNA in step (1) and step (2), measure the concentration, mix them, and inject them into sheep for fertilization In the egg cytoplasm, and then transplanted into the oviduct of female sheep of the same species after in vitro culture, it is used to produce transgenic sheep that co-knock out MSTN, ASIP, and BCO2 genes.

Among them, after sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B are mixed with Cas9mRNA, the final concentration is Cas9mRNA 20ng/μL, sgRNA-1M 5ng/μL, sgRNA-2M 5ng/μL , sgRNA-1A 5ng/μL, sgRNA-2A 5ng/μL, sgRNA-1B 5ng/μL, sgRNA-2B 5ng/μL.

The expression vector of the sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, and sgRNA-2B is pUC57-T7-gRNA, and the in vitro transcription vector of the Cas9 protein is pST1374-NLS-flag-linker- Cas9.

The specific targets are sgRNA-1M and sgRNA-2M for the second exon and the third exon of sheep MSTN, sgRNA-1A and sgRNA-2A for the fifth exon of sheep ASIP, and sgRNA-2A for the fifth exon of sheep BCO2. sgRNA-1B and sgRNA-2B of two exons.

Step 1: Construction of CRISPR/Cas9 system for MSTN, ASIP, and BCO2 genes

1. According to the sheep genome sequence in NCBI, select the second exon and the third exon of the MSTN gene in the sheep genome as target sites to design sgRNA-1M, sgRNA-2M, and the fifth exon of the ASIP gene as target sites Design sgRNA-1A, sgRNA-2A, and the second exon of the BCO2 gene as the target site to design sgRNA-1B, sgRNA-2B. 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 the pUC57-T7-gRNA vector containing specific sgRNA sequences: (1) Design and synthesize sgRNA-1M and sgRNA-2M that recognize the second exon and the third exon of the MSTN gene, and sgRNA-2M that recognize the ASIP gene sgRNA-1A and sgRNA-2A of the fifth exon, sgRNA-1B and sgRNA-2B of the second exon of the BCO2 gene; (2) synthesized sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, and sgRNA-2B paired oligonucleotides were annealed in vitro; (3) sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, and sgRNA-2B were passed through The BsaI site was digested and ligated, and inserted into pUC57-T7-gRNA, named pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1A, pUC57-T7-sgRNA -2A, pUC57-T7-sgRNA-1B, pUC57-T7-sgRNA-2B;

The CRISPR/Cas9 system for MSTN, ASIP, and BCO2 genes are: in vitro transcription vectors pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1A, pUC57-T7-sgRNA-2A, pUC57-T7-sgRNA-1B, pUC57-T7-sgRNA-2B, and pST1374-NLS-flag-linker-Cas9.

Step 2: In vitro transcription

Using the constructed vectors pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1A, pUC57-T7-sgRNA-2A, pUC57-T7-sgRNA-1B, pUC57-T7-sgRNA -2B and Cas9 mRNA in vitro transcription vector pST1374-NLS-flag-linker-Cas9 for in vitro transcription mediated by T7 promoter. (1) pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1A, pUC57-T7-sgRNA-2A, pUC57-T7-sgRNA-1B, pUC57-T7-sgRNA- 2B was linearized with Dra I, and pST1374-NLS-flag-linker-Cas9 was linearized with Age1; (2) Use the MEGAshortscript kit (Ambion) kit to perform pUC57-T7-sgRNA-1M and pUC57-T7-sgRNA according to the instructions -2M, pUC57-T7-sgRNA-1A, pUC57-T7-sgRNA-2A, pUC57-T7-sgRNA-1B, pUC57-T7-sgRNA-2B, pST1374-NLS-flag-linker-Cas9 in vitro transcription; (3 ) using the MEGAClear kit (Ambion) kit, according to the instructions for pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, pUC57-T7-sgRNA-1A, pUC57-T7-sgRNA-2A, pUC57-T7-sgRNA - Purification of 1B, pUC57-T7-sgRNA-2B, Cas9 mRNA.

Step 3: Using the CRISPR/Cas9 system for MSTN, ASIP, and BCO2 genes to produce gene-targeted sheep that co-knock out MSTN, ASIP, and BCO2 genes

1. Pronuclear injection and embryo transfer

Embryos at the single-cell stage were surgically collected from donor ewes that had naturally mated after estrus synchronization, and the premixed sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA -1B, sgRNA-2B, Cas9mRNA mixture (the final concentration of Cas9mRNA after mixing is 20ng/μL, sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B are 5ng/μL each) injected into in the cytoplasm of sheep 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 the recipient sheep to produce co-knockout of MSTN, ASIP, and BCO2 genes gene targeting sheep.

2. Identification of gene-targeted sheep with co-knockout of MSTN, ASIP and BCO2 genes

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, the primers for the second exon and the third exon of sheep MSTN, the fifth exon of ASIP, and the second exon of BCO2 were amplified with the sequences shown in Table 3, and the obtained PCR The product was detected by agarose gel electrophoresis and the product system was recovered. The recovered PCR product was digested with T7EN1 enzyme, and then electrophoresis was detected after digestion. The detection results showed that two or more bands may indicate successful gene targeting; The recovered PCR products were sent for sequencing and sequence analysis, combined with T7EN1 enzyme digestion results and sequencing results to determine positive individuals; the PCR products of positive individuals were cloned into T vectors, and after transformation, positive clones were picked for sequencing again. One step is to determine the positive individuals with successful gene knockout and the base change mode in the positive individuals.

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 kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly, it is characterised in that Comprise the following steps：

(1) build selectively targeted：MSTN Second Exons and the 3rd extron, the extrons of ASIP the 5th, BCO2 second are outer aobvious The sgRNA of son in-vitro transcription carrier；Obtained by in-vitro transcription for MSTN Second Exons and the 3rd extron SgRNA-1M, sgRNA-2M, it is outer aobvious for sheep BCO2 second for sgRNA-1A, sgRNA-2A of the extrons of ASIP the 5th SgRNA-1B, sgRNA-2B of son；

(2) the in-vitro transcription carrier of in-vitro transcription Cas9 albumen, obtains Cas9 mRNA；

(3) by the sgRNA-1M of step (1) and step (2), sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, SgRNA-2B and Cas9 mRNA survey concentration after purification, then by sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, SgRNA-1B, sgRNA-2B are mixed with Cas9 mRNA, are injected into sheep zygotes cytoplasm, are then moved after in vitro culture It is implanted into female sheep fallopian tubal of the same race, for producing the common Transgenic Sheep for knocking out MSTN, ASIP, BCO2 gene.

2. one kind according to claim 1 knocks out sheep MSTN, ASIP, BCO2 base jointly using CRISPR/Cas9 systems The method of cause, it is characterised in that sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B with Cas9 mRNA mixing after, final concentration of Cas9 mRNA 20ng/ μ L, sgRNA-1M 5ng/ μ L, sgRNA-2M 5ng/ μ L, sgRNA-1A 5ng/μL、sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。

3. one kind according to claim 1 knocks out sheep MSTN, ASIP, BCO2 base jointly using CRISPR/Cas9 systems The method of cause, it is characterised in that described sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B Expression vector be pUC57-T7-gRNA, the in-vitro transcription carrier of Cas9 albumen is pST1374-NLS-flag-linker- Cas9。

4. one kind according to claim 1 knocks out sheep MSTN, ASIP, BCO2 base jointly using CRISPR/Cas9 systems The method of cause, it is characterised in that the selectively targeted sgRNA-1M for sheep MSTN Second Exons and the 3rd extron, SgRNA-2M, for sgRNA-1A, sgRNA-2A of the extrons of sheep ASIP the 5th, for sheep BCO2 exon 2s sgRNA-1B、sgRNA-2B。