A kind of mammalian genome modification method

A modification method, mammalian technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic material, microorganisms, etc., to achieve the effect of increasing specificity

Inactive Publication Date: 2020-05-26
青岛市畜牧兽医研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, subsequent studies have shown that Cas9 has obvious off-target effects (High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology. Fu et al, 2013; High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity. Nature Biotechnology. Pattanayak et al, 2013)

Method used

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  • A kind of mammalian genome modification method
  • A kind of mammalian genome modification method
  • A kind of mammalian genome modification method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The construction of embodiment 1 plasmid vector pcDNA-DN

[0021] Synthesize the DNA fragment DN (its sequence is shown in SEQ ID NO.1), and clone it into the multiple cloning site of the pcDNA3.1(+) vector (purchased from Invitrogen, product number V79020) by BamHI and EcoRI double enzyme digestion to obtain the plasmid vector pcDNA-DN. Through sequencing verification, it is confirmed that its sequence information is shown in SEQ ID NO.2, and the sequence of the forward sequencing primer pcDNA3.1-F used is shown in SEQ ID NO.3; the sequence of the reverse sequencing primer pcDNA3.1-R is shown in Shown in SEQ ID NO.4.

[0022] pcDNA3.1-F: CTAGAGAACCCACTGCTTAC, pcDNA3.1-R: TAGAAGGCACAGTCGAGG;

Embodiment 2

[0023] Example 2 Verification of the gene modification efficiency of the genome modification method in the human cell line 293T

[0024] Human cell line 293T was purchased from Shanghai Cell Bank, Chinese Academy of Sciences, catalog number: SCSP-502.

[0025] The first exon sequence of human MSH2 gene (its sequence is shown in SEQ ID NO.5):

[0026]

[0027] Select the 120-180bp segment of the above sequence as the target, design and synthesize two DNA oligonucleotides MSH2TF and MSH2TR (the sequence is shown in SEQ ID NO.6-7) for this target, these two oligonucleotides Complementary pairing with the antisense strand of the 131-153bp region of the above sequence and the sense strand of the 161-180bp region (except the last base at the 3' end), respectively, as follows:

[0028] MSH2TF: GGTGGAGCCGAAGGAGACGCTGC C , MSH2TR: AGCCGACCTCGGCCGCGCTC G ;

[0029] MSH2TF, MSH2TR and pcDNA-DN vector were transfected into human cell line 293T according to the ratio of 1:1:5. After...

Embodiment 3

[0031] Example 3 Verification of the gene modification efficiency of the genome modification method in the mouse cell line NIH / 3T3

[0032] The mouse cell line NIH / 3T3 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-515.

[0033] The first exon sequence (its sequence is shown in SEQ ID NO.10) of mouse Tada1 gene is as follows:

[0034] 1 agagccgagc cgagccgagc cgagcggagc cgagccgagc cgagcggagc cgagccgagc

[0035] 61 cgagccgagc cgaaccgagc cgagccgagc cgaaccgagc cgagccgagc cgagtggaat

[0036] 121 cgagtcgagt cgagcctcca gcgtccggcg cgcaggcctt ccgccgcgtt gatctttcgg

[0037] 181 ttgctggtgg ccgtgggccg cgcggtctac ggtcgggctg aaagacgcgc gctgcaatgg

[0038]241 cgacctttgt gagcgagctg gaggcagcca agaagaactt gagcgaggcg ctgggggaca

[0039] 301acgtgaaaca

[0040] Select the 200-280bp segment of the above sequence as the target of genetic modification, design and synthesize two DNA oligonucleotides Tada1TF and Tada1TR (its sequence is shown in ...

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Abstract

The invention provides a modification method of the mammal genome, belonging to the technical field of genetic engineering. According to the modification method, the target gene DNA sequence of the specific area on the mammal genome is selected according to the research purpose, a pair of single-stranded DNA oligonucleotides for identifying the target gene DNA sequence is designed, the pair of single-stranded DNA oligonucleotides and a plasmid vector pcDNA-DN constructed by adopting the method provided by the invention are used for cotransfection of the mammalian cells at the mass ratio of (1-2): (1-2): (5-20), after 48h, the genome DNA is extracted, the PCR amplification is carried out, the clone sequencing is carried out, and the mutation ratio of the target region sequence is detected. Compared with the prior art, the modification method has the advantages that by synthesizing a pair of short-chain guide DNA, nuclease X is guided to cut the specific gene of the mammal in the cell or individual level, so that the purpose of knocking out or modifying one certain gene is achieved, thus the functions of the genes are analyzed, and a gene mutation pool is constructed. The modification method has the beneficial effects that the total length of the guide DNA is 40nt or longer, and the specificity of genome modification is greatly increased.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a mammalian genome modification method. Background technique [0002] ZFN, TALEN and CRISPR / Cas9 targeting technologies are several genome modification technologies that are relatively mature in research. (CRISPR) / CRISPR-associated (Cas) is a continuously evolving immune defense mechanism in bacteria and archaea. CRISPR / Cas9 uses a small RNA to recognize and cut DNA to degrade foreign nucleic acid molecules. Cong et al. (MultiplexGenome Engineering Using CRISPR / Cas Systems.Science.2013) and Mali et al. (RNA-guided human genome engineering via Cas9.Science.2013) proved that the Cas9 system can be effective in 293T, K562, iPS and other cells. Targeted enzyme digestion, non-homologous recombination (NHEJ), homologous recombination (HR) efficiency is between 3-25%, which is equivalent to TALEN enzyme digestion. They also demonstrated that multiple targets c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/87
CPCC12N15/66C12N15/85C12N15/87C12N2800/107C12N2810/10
Inventor 李和刚杨培培李培培张宝珣刘开东郝小静包汉勋孟德坤房志远苗刚张明郭成玉
Owner 青岛市畜牧兽医研究所
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