Abstract
The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.
Highlights
3" Elizabeth Culp1, Cory Richman1, Devika Sharanya and Bhagwati P Gupta* 4" Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, 5" Canada
Our approach allows for the generation of loss-of-function 8" mutations in C. briggsae genes thereby facilitating a comparative study of gene function between 9" nematodes
We describe a methodology for using the CRISPR/Cas9 system to 15" generate mutations via non-homologous end joining in the nematode Caenorhabditis briggsae, a 16" sister species of C. elegans
Summary
Our screens recovered worms with unexpected phenotypes, e.g., Dpy in. Sequencing of these worms revealed no disruption in targeted genes, 7" raising the possibility of off-target effects of CRISPR/Cas. 11" significantly enhance the efficiency of targeted mutations in C. elegans [21](23). Mutations in Cbr-unc-119 with Unc phenotype were recovered at a. 2" frequency of 11.1% (Tables 1 and 2) Another sgRNA for Cbr-unc-119 that lacked 3’. 3" GG motif did not give rise to any mutation (Table 1). 4" homolog [26], the 3’GG motif sgRNA resulted in a disruption efficiency of 9.5% The enhanced efficiency of the 3’GG motif sgRNA sites for these two genes suggests that. 6" such an approach in C. briggsae could improve the frequency of targeted mutations in genes of
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