CRISPR / su cas12a2-based gene editing system and application thereof

By using the CRISPR/SuCas12a2 gene editing system, combined with λ-Red recombinase and optimized gRNA length, efficient gene editing in multiple microbial species has been achieved, solving the problems of high lethality and immune escape in existing systems, and increasing the number of transformants and editing efficiency.

CN122588089APending Publication Date: 2026-08-18UNIV OF SCI & TECH OF CHINA
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Patent Information

Application Number
CN202610728702.4
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-05-25
Publication Date
2026-08-18

AI Technical Summary

Technical Problem

Existing CRISPR-Cas systems suffer from high lethality, low transformant numbers, and immune escape issues in microbial gene editing, making them difficult to apply efficiently across multiple species.

Method used

The gene editing system based on CRISPR/SuCas12a2 was used, which combined the SuCas12a2 protein controlled by the arabinose promoter, the crRNA elongation controlled by the T7 promoter, λ-Red recombinase and homology repair fragment, and optimized the gRNA length to 30 bp. Gene editing was performed using dual plasmid and single plasmid systems, and screening was conducted through abortion infection targeting RNA.

Benefits of technology

It improves gene editing efficiency, increases the number of transformants, reduces the probability of immune escape, and achieves efficient gene knockout and replacement in Escherichia coli, Shewanella, and Bacillus subtilis, producing 10³ to 10⁴ times the number of transformants, with broad spectrum and low cytotoxicity.

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Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a CRISPR / SuCas12a2-based gene editing system and application thereof. The present application constructs a high-efficiency counter-selection recombination editing tool by exploring the counter-selection potential of CRISPR-SuCas12a2 and combining it with lambda recombinase. The method can complete high-efficiency recombination editing of genes through a double-plasmid and single-plasmid system carrying SuCas12a2 and lambda recombinase, and the efficiency is greatly improved compared with the current recombination editing method based on lambda recombinase. Compared with the previous dependence on the specific cleavage of target double-stranded DNA by CRISPR-Cas protein to promote the effect of recombination editing, the efficiency is also obviously improved, and the method has the advantages of more transformants and low probability of immune escape. The plasmid construction, recombination editing and plasmid elimination operations can be completed within one week. The above functions supplement a powerful tool for gene recombination editing.
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