Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.
The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state-of-the-art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics. This article is protected by copyright. All rights reserved.
The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.
A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.