Inducing mutations in ebony with the act-cas9 flies

Fillip Port and Simon Bullock

MRC Laboratory of Molecular Biology, Cambridge, UK

The act-cas9 and nos-cas9 transgenics were generated in a yellow1 mutant background, making targeting yellow a little complicated and not ideal as a positive control. We therefore wanted to target another visible marker and settled for ebony, as it is very easy to identify during screening. This experiment would also give some indication of the utility of the flies for targeting different loci. We in-vitro transcribed a single gRNA targeting the ebony open reading frame just after the start codon and injected it into the act-cas9 stock at a concentration of 900ng/ul. The results are summarised in the figure below.

EbonyFigure

About 10% of injected embryos gave rise to adult flies with clones of ebony mutant tissue. Note that the injected flies are homozygous for wild-type ebony, so mutant tissue is presumably the result of biallelic targeting. Approximately 25% of the flies that developed from the injected embryos passed on mutant ebony alleles to their offspring, as assessed by crossing them with Tm3 ebony/Tm6 ebony flies. Note that this rate of transmission is lower than we observed when yellow was targeted (although we did use a pool of two independent gRNAs for yellow). Of the three flies that showed germline transmission, two had visible ebony mutant clones.

More work will be required to figure out what determines the success of a CRISPR/Cas targeting experiment. Presumably both the target gene and the specific gRNA are important determinants of success. Therefore, when attempting to mutagenise a certain locus we would recommend trying several different gRNAs. This is also of particular importance when assessing if a phenotype is only due to targeting at the desired site, as it is extremely unlikely that multiple gRNAs target common off-target sites.