CRISPR/Sa-SlugCas9 gene editing system and application thereof

Abstract

The invention belongs to the technical field of gene editing, and in particular relates to a CRISPR / Sa-SlugCas9 gene editing system and application thereof. The gene editing system of the invention isa complex formed by Sa-SlugCas9 protein and sgRNA, and can accurately target DNA sequences and generate cleavage to cause double-strand breaks to DNA. The gene editing is to perform gene editing in cells or in vitro; Sa-SlugCas9 is a fusion protein, and the PAM recognition domain (PAM interacting, PI) of SaCas9 is replaced with the PAM recognition domain (SlugCas9-PI) of SlugCas9. The Sa-SlugCas9protein is small and is 1055 amino acids, and the recognized PAM sequence is simple. The Sa-SlugCas9 protein has the amino acid sequence as shown in SEQ ID NO: 1, and the sgRNA has the nucleotide sequence as shown in SEQ ID NO: 2. The invention has wide application prospects in the field of gene editing.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a CRISPR / Sa-SlugCas9 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-ho...

AI Technical Summary

Problems solved by technology

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