CRISPR-Cas9-mediated genome editing in Caenorhabditis elegans using in-house purified Cas9 protein and sgRNAs

CRISPR-Cas9-mediated genome editing has emerged as a powerful tool for research in Caenorhabditis elegans (C. elegans). This protocol describes a comprehensive workflow for producing CRISPR-Cas9 RNPs using in-house purified Cas9 protein and in vitro-transcribed single-guide RNAs (sgRNAs), followed by microinjection into the C. elegans germline to generate genome-edited animals. The protocol covers Cas9 protein expression and purification, sgRNA design, synthesis and purification, repair template preparation, RNP assembly, germline microinjection, co-CRISPR screening, and genotyping of edited progeny. The complete procedure, from Cas9 protein purification to the isolation of homozygous mutant animals, can typically be completed within 2-3 weeks. The protocol enables the efficient generation of targeted knockouts, knock-ins, and point mutations while taking advantage of the immediate activity and reduced off-target effects associated with RNP delivery. It provides a reliable and cost-effective protocol for genome engineering and functional genomic studies in C. elegans.