CN111793654B - Method for improving CRISPR/Cas 9-mediated biallelic gene mutation efficiency and application thereof - Google Patents
Method for improving CRISPR/Cas 9-mediated biallelic gene mutation efficiency and application thereof Download PDFInfo
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- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2217/00—Genetically modified animals
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
Abstract
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 Cas9 mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration ratio of the Cas9 mRNA 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.
Description
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 use, a crRNA and a 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 + 1-site nucleotide beside the PAM sequence to promote unwinding of double-stranded DNA and realize cutting of the Cas9 protein on 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, Cas9 mRNA 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 Cas9 mRNA 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 Cas9 mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration ratio of the Cas9 mRNA to the sgRNA is 1: 10-1: 20.
to obtain better biallelic mutation efficiency, the molar concentration ratio of Cas9 mRNA 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 Cas9 mRNA 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 Cas9 mRNA 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 Cas9 mRNA and sgRNA can be selected within the injection volume range allowed in the art.
As an embodiment of the invention, Cas9 mRNA and sgRNA are simultaneously introduced by microinjection, and the molar concentration ratio of Cas9 mRNA to sgRNA is 1: 10-1: 15, the concentration of Cas9 mRNA 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 Cas9 mRNA 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 Cas9 mRNA 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 Cas9 mRNA and sgRNA into a prokaryotic embryo by microinjection;
(4) transferring Cas9 mRNA 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 very similar, so 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, Cas9 mRNA 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 a sgRNA targeting site of an 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 Cas9 mRNA and sgRNA in example 2 of the present invention, wherein A is a purified recovery band after Cas9 capping (Cas9 unliled) and poly (A) tail (Cas9 tailed) transcription; b is a purified and recovered band of the transcribed sgRNA, Cr1 and Cr2 are sgrnas for two targeting sites of the MSTN gene, and Cr3 is an sgRNA for 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.
Example 1 sgRNA design by MSTN and FGF5 gene editing
In this example, sgrnas edited by CRISPR/Cas9 gene were designed taking sheep MSTN and FGF5 genes as examples.
To achieve higher gene editing efficiency, the design principle of sgRNA is as follows: the length of the sequence complementary-paired with the genomic sequence is 20-30nt, and a PAM (NGG) sequence must be present at the 3' end of the sequence, avoiding the presence of SNPs and polyT sequences, preferably starting with the base "G".
2 targeting sites are designed aiming at the MSTN gene of the sheep and are all positioned on the third exon of the MSTN gene. The arrangement and the structure of 2 targets in an MSTN genome are shown in figure 1 and named as Cr1 and Cr2, figure 1 shows corresponding targeting sequences in the genome and PAM sections of the targets, gray sequences are crRNA sequences corresponding to sgRNAs and the targets, tracrRNAs are connected behind the sgRNAs to form the MSTN sgRNAs, and sgRNA sequences of two targeting sites are respectively shown in SEQ ID No.1 and SEQ ID No. 2.
1 targeting site is designed aiming at the FGF5 gene of sheep, the arrangement and the structure in the genome are shown in figure 2, and a grey square in figure 2 is marked as a target sequence (Cr3), and the target sequence is distributed in the middle of a beta sheet on a sense strand of the gene. The genomic sequence shown in fig. 2 includes a PAM segment, and FGF5sgRNA (sequence shown in SEQ ID No. 3) includes a crRNA sequence corresponding to the target, and a tracrRNA sequence linked thereto.
Example 2 in vitro transcription of Cas9 mRNA and sgRNA
Cas9 (the Cas9 gene sequence is shown as SEQ ID NO. 4) containing a T7 promoter and a sgRNA sequence are respectively amplified by adopting Q5 high-fidelity DNA polymerase, and an in-vitro transcription template is obtained after purification. T7-Cas9 was transcribed with the mMESSAGE mMachine T7Ultra Kit, and T7-sgRNA was transcribed with the MEGAShortscript Kit. The transcribed Cas9RNA becomes a single-stranded straight line after RNA-specific loading, and 2% agarose-TBE gel electrophoresis shows that Cas9 is about 4200nt after capping transcription, and the transcribed Cas9 mRNA band is clear and correct in size (as shown in a of fig. 3). The sgRNA (about 100nt) after transcription was purified and recovered, and the sgRNA was detected by agarose gel electrophoresis, and the sgRNA band was clear and correct in size (as shown in B of fig. 3).
Example 3 editing sheep MSTN and FGF5 genes using CRISPR/Cas9 system
In this example, a prokaryotic microinjection method is adopted to introduce Cas9 mRNA synthesized by in vitro transcription in example 2 and sgRNA of MSTN and FGF5 genes into a sheep prokaryotic embryo, so as to realize gene knockout of MSTN and FGF 5.
Cas9 mRNA synthesized by in vitro transcription in example 2 and sgRNA were mixed to prepare a gene-editing nucleic acid solution for microinjection. Sheep prokaryotic embryos were divided into three groups according to the difference in Cas9 mRNA and sgRNA injection ratios: (1) control group: cas9 mRNA and sgRNA molar concentration ratio is 1:2, two microinjection times are carried out, and the concentration of Cas9 mRNA and sgRNA of the first microinjection time is 5.23 × 10-9mol/l and 10.46 x 10-9mol/l, concentration of Cas9 mRNA and sgRNA of the second microinjection were 7.93 × 10, respectively-9mol/l and 15.86 x 10-9mol/l, the injection volume is about 5pL, 448 fertilized eggs are injected in total, 93 recipient ewes are transplanted, 30 days after embryo transplantation, B-ultrasonic examination is carried out, 35 recipients are pregnant, and 28 live lambs are produced in total; (2) experimental group 1: cas9 mRNA and sgRNA molar concentration ratio is 1:10, two microinjection times are carried out, and the concentration of Cas9 mRNA and sgRNA of the first microinjection time is 5.23 × 10-9mol/l and 5.23 x 10-8mol/l, concentration of Cas9 mRNA and sgRNA of the second microinjection were 7.93 × 10, respectively-9mol/l and 7.93 x 10-8mol/l, the injection volume is about 5pL, 365 fertilized eggs are injected in total, 84 recipient ewes are transplanted, 30 days after embryo transplantation, 26 recipients are pregnant through B ultrasonic examination, and 22 live lambs are produced in total; (3) experimental group 2: the molar concentration ratio of Cas9 mRNA to sgRNA is 1:15, wherein the concentration of Cas9 mRNA and sgRNA is 5.23 × 10-9mol/l and 7.85 x 10-8mol/l, the injection volume is about 5pL, 388 fertilized eggs are injected in total, 59 recipient ewes are transplanted, after 30 days of embryo transplantation, after B-ultrasonic examination, 17 recipients are pregnant, and 14 live lambs are produced in total.
And (3) extracting genomic DNA from lamb blood, performing PCR amplification on MSTN and FGF5 genes respectively, and sending PCR products to a company for sequencing. The gene mutation detection results are shown in table 1, the control group (the molar concentration ratio of Cas9 mRNA and sgRNA is 1:2) has 4 total lambs with mutation, the positive mutation rate of gene editing is 14.3% (4/28), but 4 lambs are all single allele mutation (namely, mutant and wild type sequences exist after target gene editing at the same time), and the double allele mutation rate is 0%; in experimental group 1 (the molar concentration ratio of Cas9 mRNA to sgRNA is 1:10), 4 lambs are mutated, the positive mutation rate of gene editing is 18.2% (4/22), all 4 lambs are biallelic mutation (i.e. all alleles on homologous chromosomes are mutated after target gene editing), and the biallelic mutation rate is 100%; in experimental group 2 (the molar concentration ratio of Cas9 mRNA to sgRNA is 1:15), 1 lamb generates mutation, and the mutation is biallelic, the positive mutation rate of gene editing is 7.1% (1/14), and the biallelic mutation rate is 100%.
TABLE 1 Gene editing efficiency of sheep MSTN and FGF5
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (3)
1. A method for improving CRISPR/Cas 9-mediated biallelic mutation efficiency of sheep MSTN gene and FGF5 gene, which is characterized by comprising the following steps: introducing a gene editing nucleic acid into a sheep prokaryotic-stage embryo; the gene-editing nucleic acid comprises Cas9 mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration ratio of the Cas9 mRNA to the sgRNA is 1: 10-1: 15;
the molar concentration of the Cas9 mRNA is 5-8 nmol/L;
the molar concentration of the sgRNA is 50-120 nmol/L;
cas9 mRNA and sgRNA were introduced simultaneously;
the sequences of sgRNAs of the MSTN gene are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the sequence of sgRNA of FGF5 gene is shown in SEQ ID NO. 3.
2. The method for improving the efficiency of CRISPR/Cas 9-mediated biallelic mutation of sheep MSTN gene and FGF5 gene according to claim 1, wherein the introducing is by microinjection.
3. The method for improving the efficiency of CRISPR/Cas 9-mediated biallelic mutation of the ovine MSTN gene and FGF5 gene according to claim 1 or 2, 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.
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