Adapting published protocols of gRNA synthesis and lesion detection1-2, we have used CRISPR/Cas9 mutagenesis to target more than 30 genomic loci. Most of the gRNAs we used were taken from the CRISPRscan track3 and >75% induced a degree of target disruption that we find to be sufficient for the generation of loss-of-function mutations that are germ line transmittable. However, with the exception of the highest-magnitude prediction scores, we observe no strong correlation between the magnitude of CRISPRscan’s gRNA-efficiency prediction scores and our quantification of gRNA efficiency outcomes. Thus, most listed CRISPRscan gRNAs are sufficient for mutant allele generation, but if particularly high cutting rates are needed, as may be for F0 phenotyping or homology-directed repair projects, gRNAs with the very highest CRISPRscan prediction scores may be preferable. We will present prediction-outcome correlations for our most up-to-date set of gRNAs, including correlations, where available, with scores from other gRNA design resources that employ distinct prediction algorithms.
Leveraging the ability to multiplex gRNAs in a single injection2 we are also exploring a strategy of including an extra gRNA, tyr-gRNA4, which targets the tyrosinase gene locus, in our microinjections. tyr-gRNA causes an observable loss of pigment in embryos and adults >48 hours post-fertilization and accordingly serves as an internal control for injection quality, Cas9 efficiency, and selection of F0 adults with extensive albinism, which presumably indicates they are more likely to transmit germ-line mutations. We have tested this strategy for a limited number of F0 adults, leading to successful egg production in 3 of 4 lines tested. With more tyr-gRNA-injected F0s in our pipeline, more definitive data is forthcoming, but we currently hypothesize that inclusion of tyr-gRNA facilitates our mutant-generation pipeline with no significantly adverse effects on viability or breeding.
1Varshney GK et al., (2015). Genome Res., 25(7):1030-42.
2Carrington B et al., (2015) Nucleic Acids Res., 43(22):e157
3M. A. Moreno-Mateos et al., (2015). Nature Methods 12,982–988
4L.E. Jao et al., (2013) Proc. Natl. Acad. Sci. U.S.A., 110:13904–990.