A CRISPR/Cas9-based system using dual-sgRNAs for efficient gene deletion in Mycobacterium abscessus

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Mycobacterium abscessus infections are increasing globally, posing a significant clinical challenge due to antibiotic resistance and poor treatment outcomes.
• Efficient genetic tools, particularly for large-fragment deletions, are crucial for studying M. abscessus physiology, pathogenesis, and drug resistance but are currently underdeveloped.

Purpose of the Study:

• To develop and optimize a CRISPR-Cas9-based system for efficient large-fragment deletions in Mycobacterium abscessus.
• To advance functional genomics studies and targeted investigations into M. abscessus virulence and antibiotic resistance mechanisms.

Main Methods:

• Implementation of a CRISPR/Cas9-based dual-single-guide RNA (sgRNA) system utilizing Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9).
• Systematic optimization of the modular arrangement of genetic components in Cas9/dual-sgRNA expression plasmids and refinement of their construction workflow.
• Evaluation of deletion efficiencies at various loci and correlation with target size and genomic position.

Main Results:

• Achieved efficient large-fragment knockout in M. abscessus, with deletion efficiencies exceeding 90% at specific loci and spanning up to 16.7 kilobases (kb).
• Significantly reduced cassette loss rates and enabled single-step plasmid assembly through optimized plasmid design and workflow.
• Observed that deletion efficiency was position-dependent, suggesting the influence of chromatin structure rather than target size on editing outcomes.

Conclusions:

• This study presents the first CRISPR/Cas9-based platform enabling kilobase-scale deletions in M. abscessus.
• The developed system significantly enhances the capacity for functional genomics research in M. abscessus.
• This tool facilitates targeted investigations into the complex mechanisms of virulence and antibiotic resistance in M. abscessus infections.