CRISPR/Sa-SeqCas9 gene editing system and application thereof

Abstract

The invention belongs to the technical field of gene editing, and particularly discloses a CRISPR / Sa-SeqCas9 gene editing system and application thereof. The gene editing system is a complex formed bySa-SeqCas9 protein and sgRNA, a DNA sequence can be precisely targeted, and cutting is also conducted to cause DNA double strain break damage; the gene editing is gene editing conducted in cells or on vitro; and an Sa-SeqCas9 gene is a fusion protein, a PAM recognition structural domain of SaCas9 (PAM interacting, PI) is replaced by the PAM recognition structural domain of SeqCas9 (SeqCas9-PI); the Sa-SeqCas9 protein is small and has 1053 amino acids, the recognized PAM sequence is simple, the Sa-SeqCas9 protein has an amino acid sequence shown in SEQ ID NO: 1, and the sgRNA has a nucleotidesequence shown in SEQ ID NO: 2. The CRISPR / Sa-SeqCas9 gene editing system has wide application prospects in the field of the gene editing

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a CRISPR / Sa-SeqCas9 system capable of gene editing in cells and related applications thereof. Background technique [0002] CRISPR / Cas9 is an adaptive immune system evolved by bacteria and archaea to resist the invasion of foreign viruses or plasmids. In the CRISPR / Cas9 system, after crRNA (CRISPR-derived RNA), tracrRNA (trans-activating RNA) and Cas9 protein form a complex, they recognize the PAM (Protospacer Adjacent Motif) sequence of the target site, and the crRNA will form a complementarity with the target DNA sequence Structure, the Cas9 protein performs the function of cutting DNA, causing DNA breakage and damage. Wherein, tracrRNA and crRNA can be fused into a single-stranded guide RNA (singleguide RNA, sgRNA) through a linking sequence. When DNA is broken and damaged, two main DNA damage repair mechanisms in cells are responsible for repairing: non-hom...

AI Technical Summary

Problems solved by technology

However, the SpCas9 protein has 1367 amino acids, plus sgRNA and promoter, cannot be effectively packaged into AAV virus, which limits its clinical application

To overcome this problem, several small Cas9s have been invented, including SaCas9 (PAM sequence is NNGRRT); St1Cas9 (PAM sequence is NNAGAW); NmCas9 (PAM sequence is NNNNGATT); Nme2Cas9 (PAM sequence is NNNNCC); The PAM sequence is NNNNRYAC), but these Cas9 or PAM sequences are complex (there are few DNA sequences that can be targeted in the genome), or the editing efficiency is low, and it is difficult to be widely used

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