These days it seems like new papers and reviews relevant to CRISPR-Cas9 modification pop up weekly, if not daily, making it hard to keep up.
I am admittedly biased—the authors are close colleagues—but that said, a recent chapter in Methods in Enzymology from Housden et al. (2014) seems worth a look by folks interested in applying CRISPR-Cas9 or other gene editing techniques in Drosophila melanogaster or other insect species.
The chapter presents both a nice review and step-by-step protocols.
Common challenges in applying a gene editing technique include design of an appropriate short guide RNA (gRNA) and isolation of animals with events of interest from among a mixed population. Two sections in the chapter present approaches that can help and notably, could be applied to non-model insect species.
Firstly, in section 2.1 of the chapter, the authors discuss the types of decisions that should be considered in choosing a gRNA appropriate for the type of lesion or change you are interested to engineer. These include where the gRNA sequence should be relative to the specific region targeted for modification, and how to think about off-targets in the context of a genetic system for which backcrosses can be used to ‘clean up’ off targets, as compared with cultured cell systems in which that is not possible.
“Altogether, the chapter provides a nice resource of background information and protocols that should help facilitate CRISPR-Cas9 studies in Drosophila and other species.” Stephanie Mohr.
Secondly, in section 5.1, the authors describe a protocol facilitating use of a single fly wing as starting material for PCR-based analysis of CRISPR-mediated events. Researchers with a delicate touch can remove a wing for PCR analysis while still keeping the fly itself alive. This makes it possible to do molecular analysis at a stage when only a single animal carries the event of interest, saving time and resources needed to establish stocks. The approach should be applicable to any species for which a small amount of material can be collected without killing or sterilizing the animal. In addition, the approach should be useful not just for CRISPR-Cas9 follow-up but also for PCR analysis of other types of genome changes.
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