CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-Cas9) homing sequences and primers thereof, and transgenic expression vector and establishment method thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-Cas9) homing sequences and primers thereof, and a transgenic expression vector and an establishment method thereof. The CRISPR-Cas9 homing sequence sgRNA comprises SIDT1-gene-specific Target site-1 of which the target is positioned on E1 exon, and SIDT1-gene-specific Target site-2 of which the target is positioned on E2 exon, wherein the nucleotide sequence of the Target site-1 is disclosed as SEQ ID NO.8, and the nucleotide sequence of the Target site-2 is disclosed as SEQ ID NO.9. The CRISPR-Cas9 transgenic expression vector is formed by connecting the Cas9 homing sequence Target site-1 and Target site-2 to the BsmB I and Bbs I enzyme digestion sites of a recombinant shuttle Cas9 tool plasmid. The recombinant shuttle Cas9 tool plasmid can be quickly assembled with homing sequences of the two target sites of the genome, thereby packaging the recombinant adenovirus vector specifically for the two target sites; and the recombinant shuttle Cas9 tool plasmid can independently complete the editing of the large-segment genome without dependence on the synergistic actions. The establishment process is simple and quick, and has high efficiency for performing the genome large-segment editing function.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a CRISPR-Cas9 guide sequence primer, a transgene expression vector (especially a double-promoter CRISPR-Cas9 transgene vector based on a replication-defective adenovirus) and a construction method thereof. Background technique [0002] Genome editing can be realized by DNA fragment deletion, chromosome inversion, DNA fragment insertion, etc. It is one of the important means to study gene function, and it can also be used for the treatment of human genetic diseases, the treatment of virus integration-related diseases and animal models Therefore, this type of technology has become a research hotspot in modern molecular biology. The early gene targeting technology based only on homologous recombination was extremely inefficient and its application was limited. With the emergence of artificial endonuclease (engineered endonuclease, EEN), this situation has been comple...

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Problems solved by technology

[0005] However, the recognition of targets by ZFNs and TALENs mainly depends on the recognition of nucleic acids by DNA-binding proteins. A zinc finger protein (structural unit) in ZFNs recognizes three base sequences, while an RVD in TALENs recognizes one base. In order to ensure specificity Generally, the length of the target is 18-20bp. Therefore, when constructing ZFN or TALEN, it is necessary to arrange and combine zinc finger protein units or RVD according to the sequence of the target. There are many fragments to be spliced, cumbersome operations, and a long preparation period. long, labor-intensive and cost-intensive

In addition, the vectors completed with a lot of labor and expense can only be used for gene editing at one recognition site. If large fragment gene editing is to be realized, vectors targeting two sites must be constructed at the same time, and these vectors need to be combined in the future. Transferring into the target sample and relying on synergy to complete gene editing at a specific site is difficult, costly, inefficient, and takes a long time

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