Elsevier

New Biotechnology

Volume 66, 25 January 2022, Pages 61-69
New Biotechnology

CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum

https://doi.org/10.1016/j.nbt.2021.10.003Get rights and content
Under a Creative Commons license
open access

Highlights

  • CRISPR-Cas12a could mediate precise genome integration with the pNIL/pGOAL system.

  • CRISPR-Cas12a was remarkable in facilitating one-step deletion of DNA with 1–24 Kb.

  • Cas12a had little effect on Mycolicibacterium neoaurum cell activity.

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.

Abbreviations

CRISPR
clustered regularly interspaced short palindromic repeats
NHEJ
nonhomologous end-joining
HR
homologous recombination
SCO
single crossover
DCO
double crossover
DSBs
double-strand breaks
crRNA
CRISPR RNA
C.F.U
colony-forming units
HBC
22-hydroxy-23 24-bisnorchol-4-ene-3-one
9−OHAD
9α-hydroxyandrost-4-ene-317-dione

Keywords

CRISPR-Cas12a
Genome editing
Mycolicibacterium neoaurum
NHEJ
Gene integration

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