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

Aniek S. Meijers, Ran Troost, Roy Ummels, Janneke Maaskant, Alexander Speer, Sergey Nejentsev, Wilbert Bitter, Coenraad P. Kuijl*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Article number101983
Publication statusPublished - Sept 2020


  • CRISPR-Cas9 system
  • Genome editing
  • Indels
  • Mycobacterium marinum
  • Mycobacterium tuberculosis


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