Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9 release_4yf6f4ms3fba3bpwjcvw5cz4q4

by Aniek S. Meijers, Ran Troost, Roy Ummels, Janneke Maaskant, Alexander Speer, Sergey Nejentsev, Wilbert Bitter, Coen Kuijl

Published in Tuberculosis by Elsevier BV.

2020   Volume 124, p101983

Abstract

The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.
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