CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum

Ke Liu; Yang Gao; Zhen-Hai Li; Min Liu; Feng-Qing Wang; Dong-Zhi Wei

doi:10.1016/j.nbt.2021.10.003

CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum

N Biotechnol. 2022 Jan 25:66:61-69. doi: 10.1016/j.nbt.2021.10.003. Epub 2021 Oct 12.

Authors

Ke Liu¹, Yang Gao², Zhen-Hai Li³, Min Liu⁴, Feng-Qing Wang⁵, Dong-Zhi Wei⁶

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. Electronic address: y12190016@mail.ecust.edu.cn.
² State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. Electronic address: gaoyang3013@163.com.
³ State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. Electronic address: zhenhai_li@126.com.
⁴ State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. Electronic address: lmin@ecust.edu.cn.
⁵ State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. Electronic address: fqwang@ecust.edu.cn.
⁶ State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. Electronic address: dzhwei@ecust.edu.cn.

PMID: 34653700
DOI: 10.1016/j.nbt.2021.10.003

Abstract

Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.

Keywords: CRISPR-Cas12a; Gene integration; Genome editing; Mycolicibacterium neoaurum; NHEJ.

MeSH terms

CRISPR-Cas Systems* / genetics
DNA End-Joining Repair
Gene Editing*
Mycobacteriaceae / genetics*
Plasmids

Supplementary concepts

Mycolicibacterium neoaurum