Xu et al. review the recent history of using CRISPR/Cas9-based technologies in Drosophila melanogaster.
Within the last two years there have been numerous publications describing the use of CRISPR/Cas9-based technologies in Drosophila melanogaster. These last two years have been a time of shaking out the technology and exploring various aspects of the technology such as methods of delivery, methods for detecting mutations and factors affecting efficiency.
Xu et al. look at each of these topics.
Almost every possible method for delivering Cas9 and sgRNAs to developing embryos of D. melanogaster has been tried and reported. Xu et al. discuss injection of mRNA and sgRNA directly, expression of each from plasmids and finally, expression from integrated transgenes. For general applicability and simplicity Xu et al. feel that direct injection of RNA is the method of choice and is applicable to species other than D. melanogaster. This approach is certainly versatile but it may also be worth considering direct injection of purified Cas9. Some, including those in my lab, are finding direct injection of Cas9 protein to be very effective in non-drosophilid species.
Xu et al. discuss various mutant detection strategies and offer some words of wisdom,
“Although screening for successful gene editing events is one of the last steps in applying the CRISPR system, it should be considered thoroughly even before designing the sgRNAs, since it can easily become the bottleneck in mutagenesis practice”
Three approaches to detecting mutations are mentioned including sequencing, high resolution melt analysis (HRMA), and restriction fragment length polymorphism (RFLP) but do not mention heteroduplex cleavage assays. Nonetheless their discussion is helpful and they address issues associated with promoting homologous recombination.
Xu et al. also review what has been determined about designing sgRNA and choosing target sites in D. melanogaster. They rightfully warn users that this step is critical and they provide some specific guidance and point to a number of online resources that can help with these problems. While they acknowledge that target sequences are usually 20 nt they recount some of their own experiences with targets ranging from 17-22 nt. Targets of 18 nt are as efficient as those of 20 nt and, according to the authors, have the potential for lower off-target effects. Furthermore, they refer to their earlier work that showed that a high GC content in the seed region and in particular the 6 nt proximal to the PAM are more likely to result in high mutagenesis efficiency.
The development and use of CRISPR/Cas9 has been occurring at a frantic pace and keeping up on all of the latest developments is an ongoing challenge. The efforts in Drosophila melanogaster alone have been substantial.
Xu et al. bring much of that work together and also provide a good aggregation of references that will be helpful to those working with or contemplating working with this powerful and versatile genetic technology.
Xu, J., Ren, X., Sun, J., Wang, X., Qiao, H.-H., Xu, B.-W., Liu, L.-P., Ni, J.-Q.,
A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila, Journal of Genetics and Genomics (2015), doi: 10.1016/j.jgg.2015.02.007.