crispr/sa-slugcas9 gene editing system and its application

Abstract

The invention belongs to the technical field of gene editing, in particular to a CRISPR / Sa‑SlugCas9 Gene editing systems and their applications. The gene editing system of the present invention is Sa‑SlugCas9 The complex formed by the protein and the sgRNA can precisely target the DNA sequence and produce cutting to cause double-strand break damage to the DNA; the gene editing is gene editing in cells or in vitro; Sa‑SlugCas9 For the fusion protein, replace the PAM recognition domain (PAM interacting, PI) of SaCas9 with SlugCas9 The PAM recognition domain (SlugCas9‑PI). Sa‑SlugCas9 The protein is small, 1055 amino acids, and the recognized PAM sequence is simple. Sa‑ SlugCas9 The protein has the amino acid sequence shown in SEQ ID NO:1, and the sgRNA has the nucleotide sequence shown in SEQ ID NO:2. The invention has broad application prospects in the field of gene editing.

Description

technical field [0001] The invention belongs to the technical field of gene editing, in particular to a CRISPR / Sa-SlugCas9 system capable of performing gene editing in cells and related applications thereof. Background technique [0002] CRISPR / Cas9 is an adaptive immune system evolved by bacteria and archaea to defend against foreign virus or plasmid invasion. In the CRISPR / Cas9 system, after crRNA (CRISPR-derived RNA), tracrRNA (trans-activating RNA) and Cas9 protein form a complex, the PAM (Protospacer Adjacent Motif) sequence of the target site is recognized, and the crRNA will be complementary to the target DNA sequence. Structure, Cas9 protein performs the function of cutting DNA, causing DNA breakage damage. Among them, tracrRNA and crRNA can be fused to form a single-stranded guide RNA (single guideRNA, sgRNA) through a connecting sequence. When DNA is damaged by breakage, two major DNA damage repair mechanisms in cells are responsible for the repair: non-homologou...

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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