Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in C. elegans.
The Caenorhabditis elegans Gene Knockout (KO) Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately15,000 ORFs have associated putative null alleles. A directed approach using CRISPR/Cas9 methodology is the most promising technique to complete the task. While there has been substantial success in using CRISPR/Cas9 in C. elegans, there has been little emphasis on optimizing the method for generating large insertions/deletions in this organism. To enhance the efficiency of using CRISPR/Cas9 to generate gene knockouts in C. elegans we have developed an online species-specific guide RNA selection tool (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust, efficient and effective protocol for generating deletions. Debate and speculation in the larger scientific community about off- target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude this modified protocol does not generate off-target events to any significant degree in C. elegans.