Optimizing guide RNA selection and CRISPR-Cas9 methodology for efficiently generating deletions in C. elegans and minimizing off-target events.

The C. elegans ko consortium is tasked with obtaining null mutations in all 20,000 plus open reading frames (ORF's) of this organism. To date, about 15,000 ORF's have associated deletion or nonsense alleles. To complete this task it is clear the best way forward is to use a directed approach as exemplified by CRISPR-Cas9 methodology. However, while there has been plenty of success in using this methodology in C. elegans there has been little emphasis on optimizing the protocol. To enhance the efficiency of using this protocol for C. elegans we have developed a species-specific guide RNA site (http://genome.sfu.ca/crispr/search.html). When coupled to previously developed selection vectors, optimized homology arm length, and the use of purified Cas9 protein we demonstrate a robust efficient and effective protocol. As there has been much debate and speculation in the larger scientific community about off-target effects due to Cas9 non-specific cutting we have investigated through whole genome sequencing the occurrence of single nucleotide variants or 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 in this organism.