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Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9
Aniek S Meijers1, Ran Troost1, Roy Ummels2
1Department of Medical Microbiology and Infection Control, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, Netherlands.
Tuberculosis (Edinburgh, Scotland)
|August 24, 2020
Summary
Researchers adapted CRISPR interference for precise genome editing in Mycobacterium tuberculosis and Mycobacterium marinum. This new tool enables efficient DNA breaks and gene modifications without off-target effects, accelerating research on these pathogens.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Advancements in molecular tools have enhanced genetic engineering of pathogenic mycobacteria.
- CRISPR-Cas9 systems from Streptococcus pyogenes and Streptococcus thermophilus have been applied for gene editing and transcriptional regulation in mycobacteria.
Purpose of the Study:
- To convert CRISPR interference into an efficient genome editing tool for mycobacteria.
- To demonstrate the functionality and efficiency of the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) system in Mycobacterium marinum and Mycobacterium tuberculosis.
Main Methods:
- Adaptation of CRISPR interference for genome editing in mycobacteria.
- Utilizing Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) system.
- Employing dual single-guide RNAs (sgRNAs) for simultaneous modifications.
Main Results:
- Sth1Cas9 system is functional in Mycobacterium marinum and Mycobacterium tuberculosis.
- Highly efficient and precise DNA breaks and indel formation were achieved.
- No off-target effects were observed.
- Simultaneous generation of two indels and specific deletions were possible using dual sgRNAs.
Conclusions:
- CRISPR interference is an effective genome editing tool for mycobacteria.
- The Sth1Cas9 system offers a powerful, precise, and efficient method for genetic manipulation in M. tuberculosis and M. marinum.
- This single-step gene editing approach will accelerate research into M. tuberculosis, a significant global pathogen.
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