Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CRISPR RNA-guided Cas9 nuclease is a versatile tool for genome editing across diverse organisms.
• Targeted gene modification is crucial for understanding gene function and biological processes.

Purpose of the Study:

• To investigate the efficacy of dual single-guide RNA (sgRNA)-guided Cas9 nuclease for generating knockout mutants in C. elegans.
• To determine the maximum size of chromosomal intervals that can be deleted using this dual sgRNA/Cas9 strategy.

Main Methods:

• Co-injection of C. elegans with dual sgRNAs and Cas9 nuclease.
• Utilizing the CRISPR/Cas9 system to target protein-coding genes, noncoding genes, and repetitive sequences.
• Employing PCR and agarose electrophoresis for screening large deletions.

Main Results:

• Dual sgRNA-guided Cas9 successfully generated knockout mutants by deleting intervals between sgRNAs.
• Demonstrated the deletion of at least a 24 kb chromosome segment using this method.
• Facilitated efficient mutant screening through the detection of large deletions.

Conclusions:

• The dual sgRNA/Cas9 system offers a powerful and efficient platform for generating gene knockout mutants in C. elegans.
• Large chromosomal segment deletions simplify the screening process for desired mutants.
• Simultaneous editing of multiple genomic sites using a single CRISPR array may lead to off-target deletions.