The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/ Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (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 and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning 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 that this modified protocol does not generate off-target events to any significant degree in C. elegans. We did, however, observe a number of nonspecific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events.
Bibliographical noteFunding Information:
Dr. John Calarco (University of Toronto) and Dr. Geraldine Seydoux (Johns Hopkins University) kindly provided us with reagents and much valued advice as we refined the CRISPR/Cas9 system in our laboratory. We also thank Mei Zhen and Julie Claycomb, for their critical support during the early stages of these studies. This work was funded by CIHR grant PJT-148549 to DGM, CIHR grant MOP 93719 and NSERC grant 312498 to HH, NIH grant 5P40OD010440 to AR and NIH grant 5R240D023041 to AR, Paul Sternberg, Geraldine Seydoux and DGM.
© 2019 by the Genetics Society of America.
- C. elegans