Additional CRISPR/Cas9 expression vector and construction method and application thereof

Abstract

The invention relates to an additional CRISPR/Cas9 expression vector and a construction method and application thereof, and belongs to the field of cell engineering. In order to solve the problem thatan existing CRISPR/Cas expression system is easily integrated into a genome of a host cell and accordingly the sequence in the genome is destroyed, the invention provides the additional CRISPR/Cas9 expression vector. The vector is a CRISPR/Cas9 expression vector containing an S/MAR sequence. The nucleotide of the S/MAR sequence is as shown in SEQ ID NO:1, and the S/MAR sequence is located betweena coding region sequence of encoded protein in the vector and a bGH poly(A) sequence. The additional CRISPR/Cas9 expression vector can be used for expressing Cas9 protein and single-stranded guide RNA outside a chromosome, and can be used for gene knockout research.

Description

Technical field [0001] The invention belongs to the technical field of cell engineering, and specifically relates to an additional CRISPR / Cas9 expression vector and a construction method and application thereof. Background technique [0002] The CRISPR / Cas9 system, as an emerging powerful gene editing and genetic screening tool, has broad application prospects in the establishment of disease models and gene therapy in biomedical fields through gene editing. The CRISPR / Cas9 system mainly includes two members in the process of performing gene editing functions, Cas9 protein and single-stranded guide RNA (sigle-guide RNA, sgRNA). Among them, Cas9 protein and single-stranded guide RNA are mainly obtained by transcription and expression of expression vectors in tissue cells. Commonly used expression vectors are generally retroviral expression vectors or plasmid expression vectors. However, the CRISPR / Cas9 system still faces some technical difficulties in the actual application proces...

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problem that the existing CRISPR / Cas expression system is easy to integrate into the host cell genome and destroy the sequence in the genome, an episomal CRISPR / Cas9 expression vector is provided, which refers to a CRISPR / Cas9 expression vector containing S / MAR sequence, The S / MAR sequence, its nucleotide is shown in SEQ ID NO: 1; the S / MAR sequence is located between the coding region sequence capable of encoding protein and the bGH poly(A) sequence in the vector

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