阅读说明：本技术 一种提高CRISPR/Cas9介导的双等位基因突变效率的方法及其应用 (Method for improving CRISPR/Cas 9-mediated biallelic gene mutation efficiency and application thereof ) 是由 连正兴 李岩 邓守龙 刘国世 连玲 于 2019-04-08 设计创作，主要内容包括：本发明涉及基因工程和分子遗传育种技术领域,具体涉及一种提高CRISPR/Cas9介导的双等位基因突变效率的方法及其应用。本发明提供的CRISPR/Cas9基因编辑方法包括向原核期胚胎中导入基因编辑核酸的步骤,所述基因编辑核酸包括Cas9 mRNA和sgRNA；所述基因编辑核酸中,所述Cas9 mRNA和sgRNA的摩尔浓度比例为1：10～1：20。本发明提供的基因编辑方法,显著提高了双等位基因突变的效率,进而有效降低了基因编辑嵌合个体的比例,提高了双等位基因突变个体的比例,对于目标性状动物的构建和遗传育种具有重要的应用价值。(The invention relates to the technical field of genetic engineering and molecular genetic breeding, in particular to a method for improving CRISPR/Cas 9-mediated biallelic gene mutation efficiency and application thereof. The CRISPR/Cas9 gene editing method provided by the invention comprises the steps of introducing gene editing nucleic acid into a prokaryotic embryo, wherein the gene editing nucleic acid comprises Cas9mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration ratio of the Cas9mRNA to the sgRNA is 1: 10-1: 20. the gene editing method provided by the invention obviously improves the efficiency of biallelic gene mutation, further effectively reduces the proportion of gene editing chimeric individuals, improves the proportion of biallelic gene mutant individuals, and has important application value for construction and genetic breeding of target character animals.)

1. A method of increasing the efficiency of CRISPR/Cas 9-mediated biallelic mutation, comprising: introducing a gene-editing nucleic acid into a prokaryotic embryo; the gene-editing nucleic acid comprises Cas9mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration ratio of the Cas9mRNA to the sgRNA is 1: 10-1: 20.

2. the gene editing method of claim 1, wherein the molar concentration ratio of Cas9mRNA to sgRNA is 1: 10-1: 15.

3. the gene editing method of claim 1 or 2, wherein the molar concentration of the Cas9mRNA is 5-8 nmol/L;

and/or the presence of a gas in the gas,

the molar concentration of the sgRNA is 50-120 nmol/L.

4. A gene editing method according to any one of claims 1 to 3, wherein the introduction of Cas9mRNA and sgRNA is simultaneous.

5. The method of gene editing according to any one of claims 1 to 4, wherein the introduction is performed by microinjection.

6. A gene editing method according to any one of claims 1 to 5, comprising the steps of:

(1) synthesis of gene-editing nucleic acids;

(2) introducing a gene-editing nucleic acid into a prokaryotic embryo;

(3) and (4) identifying the individuals positive for gene editing.

7. The method for gene editing according to any one of claims 1 to 6, wherein the prokaryotic embryo is a non-human mammalian prokaryotic embryo.

8. Use of the method of gene editing according to any one of claims 1 to 7 for the preparation of a gene-edited animal or genetic breeding of an animal.

9. Use of a method of gene editing according to any one of claims 1 to 7 to increase the efficiency of biallelic mutation.

Technical Field

The invention relates to the technical field of genetic engineering and molecular genetic breeding, in particular to a method for improving CRISPR/Cas 9-mediated biallelic gene mutation efficiency and application thereof.

Background

The CRISPR/Cas9 technology is an RNA-mediated genome editing technology, and can perform accurate knockout, knock-in, replacement and the like on genes, thereby achieving the purposes of researching gene functions, removing or repairing targeted genes.

The CRISPR/Cas9 system is mainly composed of two parts: sgRNA and Cas9 protein. The sgRNA consists of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), and the crRNA includes a sequence capable of pairing with the tracrRNA to form a double-stranded RNA structure, and a sequence capable of being complementary to a target region of a target gene, and thereby recognizes the target sequence. In practical applications, crRNA and tracr RNA are often chimeric into a single-stranded guide RNA (sgRNA). When the CRISPR/Cas9 gene editing system carries out gene editing, Cas9 protein and sgRNA form a complex firstly, the complex recognizes a PAM sequence (NGG sequence) of a target gene, and Cas9 protein interacts with a + 1-site nucleotide beside the PAM sequence to promote unwinding of double-stranded DNA, so that the Cas9 protein cuts a target region. The repair modes of the genome double-stranded DNA break mainly comprise non-homologous end connection and homologous mediated double-stranded DNA repair. Non-homologous end joining is a repair with a very high error rate, with frame-shift insertions or deletions resulting in loss of gene function. Homology-mediated double-stranded DNA repair, although less than 10% is involved, can insert the desired sequence precisely at the target site, and can be used for precise site-directed editing or gene knock-in.

When the CRISPR/Cas9 system is used for gene editing of animals, Cas9mRNA translated to generate Cas9 protein and sgRNA are generally directly introduced into prokaryotic embryos by microinjection, but mosaic phenomenon exists when animal mutants are obtained. The mosaic phenomenon refers to that when the CRISPR/Cas9 system is applied to gene editing of fertilized eggs of multicellular organisms, because the fertilized eggs are divided into different blastomeres, and editing capacity and repair modes of Cas9 protein on different blastomeres may be different, chimeric individuals with edited cells and unedited cells are caused to appear, and further the gene editing efficiency of animals and the acquisition of animals with target characters are influenced. Therefore, the development of a CRISPR/Cas9 gene editing method which increases the proportion of biallelic mutant individuals and reduces the proportion of chimeric individuals is urgently needed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a method for improving CRISPR/Cas 9-mediated biallelic mutation efficiency and application thereof.

When the CRISPR/Cas9 system is used for gene editing of fertilized eggs or embryonic cells of a multicellular organism, the Cas9 system may have different editing efficiency and repair modes for different cells in the cell division process, so that the mutation efficiency of alleles is different, finally, chimeric individuals with gene editing cells and unedited cells are generated, and the gene editing efficiency of the multicellular organism and the acquisition of target character individuals with double-allele mutation are influenced. During research, the inventors found that the efficiency of biallelic gene editing can be significantly improved by increasing the molar concentration ratio of the sgRNA and Cas9mRNA that are conventionally used and introduced into cells.

First, the present invention provides a method of increasing the efficiency of CRISPR/Cas 9-mediated biallelic mutation, the method comprising the steps of introducing into a prokaryotic embryo a gene-editing nucleic acid comprising Cas9mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration ratio of the Cas9mRNA to the sgRNA is 1: 10-1: 20.

to obtain better biallelic mutation efficiency, the molar concentration ratio of Cas9mRNA and sgRNA in the gene editing nucleic acid is 1: 10-1: 15.

in order to obtain better biallelic gene mutation efficiency and simultaneously reduce the influence of introduced nucleic acid on cells as much as possible, the molar concentration of Cas9mRNA introduced into the cells is 5-8 nmol/L. The molar concentration of the sgRNA is 50-120 nmol/L.

In the present invention, the Cas9mRNA and the sgRNA are introduced simultaneously.

In the invention, the introduction is carried out by means of microinjection.

The volume of the gene-editing nucleic acid comprising Cas9mRNA and sgRNA can be selected within the injection volume range allowed in the art.

As an embodiment of the invention, Cas9mRNA and sgRNA are simultaneously introduced by microinjection, and the molar concentration ratio of Cas9mRNA to sgRNA is 1: 10-1: 15, the concentration of Cas9mRNA in the introduced gene editing nucleic acid is 5-8 nmol/L; the concentration of sgRNA is 50-120 nmol/L, and the volume of the introduced gene editing nucleic acid is 3-10 pL.

The molar concentration ratio of Cas9mRNA and sgRNA is adopted to conduct gene editing nucleic acid introduction, gene editing realized by a non-homologous end connection mode is conducted on a prokaryotic embryo, and the mutation efficiency of double alleles is remarkably improved.

Specifically, the gene editing method of the present invention comprises the steps of:

(1) synthesis of gene-editing nucleic acids;

(2) introducing a gene-editing nucleic acid into a prokaryotic embryo;

(3) and (4) identifying the individuals positive for gene editing.

In the invention, the prokaryotic embryo is a non-human mammal prokaryotic embryo.

As an embodiment of the present invention, when the prokaryotic embryo is derived from sheep, the gene editing method comprises the steps of:

(1) in vitro transcription synthesis of Cas9mRNA and sgRNA, which are gene editing nucleic acids;

(2) carrying out in-vitro insemination after superovulation of a donor sheep to obtain a prokaryotic embryo;

(3) introducing Cas9mRNA and sgRNA into a prokaryotic embryo by microinjection;

(4) transferring Cas9mRNA and sgRNA injected embryos into recipient sheep;

(5) and (3) identifying the gene editing positive individuals of the lamb individuals produced by the recipient sheep.

The principle and the action process of the CRISPR/Cas9 gene editing method in mammalian embryos are quite similar, so that the CRISPR/Cas9 gene editing method provided by the invention is suitable for all prokaryotic embryos of non-human mammals, such as: mouse, pig, cow, sheep, or monkey.

Further, the invention also provides an application of the gene editing method in any one of the following aspects:

(1) the application in preparing gene editing animals;

(2) the application in animal genetic breeding;

(3) application in improving the efficiency of biallelic gene editing.

The invention has the beneficial effects that: when the CRISPR/Cas9 system is used for gene editing of a non-human mammal prokaryotic embryo, Cas9mRNA and sgRNA with a specific molar concentration ratio are introduced into the embryo, so that the efficiency of biallelic gene mutation is obviously improved, the proportion of chimeric individuals in a gene editing progeny is effectively reduced, and the proportion of biallelic gene mutant individuals is improved; meanwhile, the whole gene editing efficiency is also improved. The gene editing method provided by the invention has important application value for preparation and genetic breeding of target character animals.

Drawings

Fig. 1 is a schematic structural diagram of two sgRNA targeting sites (Cr1 and Cr2) of an MSTN gene in example 1 of the present invention, wherein the sgRNA sequence includes a crRNA sequence corresponding to a target point and a tracrRNA sequence.

Fig. 2 is a schematic structural view of sgRNA targeting sites of FGF5 gene in example 1 of the present invention, in which the positions of boxes are sgRNA targeting positions (Cr 3); the sequences within the box are PAM segments, and the sgrnas include the crRNA sequence and tracrRNA sequence corresponding to the target.

FIG. 3 is an electrophoretogram of in vitro transcription products of Cas9mRNA and sgRNA in example 2 of the present invention, wherein A is a purified recovery band after Cas9 capping (Cas9 inactive) and poly (A) tail (Cas9 inactive) transcription; b is a purified and recovered band of the transcribed sgRNA, Cr1 and Cr2 are sgRNAs aiming at two targeting sites of the MSTN gene, and Cr3 is a sgRNA aiming at a targeting site of the FGF5 gene.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.