crispr/sa-slugcas9 gene editing system and its application

A gene editing and editing technology, applied in the field of gene editing, can solve problems such as ineffective packaging, limited application, and complex PAM sequences

CN110577969BActive Publication Date: 2022-07-22FUDAN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2022-07-22

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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.
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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...

Examples

Embodiment Construction

[0053] The present invention will be further illustrated by the following examples, but the examples do not limit the present invention in any form.

[0054] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. The experimental methods that do not specify specific conditions are usually carried out in accordance with conventional conditions or conditions suggested by the manufacturer.

[0055] In a specific embodiment, the CRISPR / Sa-SlugCas9 system provided by the present invention is a new gene editing system, method, kit and application thereof.

[0056] In a specific embodiment of the present invention, the CRISPR / Sa-SlugCas9 system can perform gene editing in cells, and the method includes the following steps:

[0057] 1. Construction of plasmid p...