Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

Abstract

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

“…The Vps39 Δ1 knockout line was generated using CRISPR/Cas9-catalyzed homology-directed repair according to the method outlined in [94]. In brief, two gRNA-expressing DNA constructs targeted near each end of the coding sequence of the Vps39 gene were injected into yw; nos-Cas9(II-attP40) embryos expressing Cas9, together with a donor DNA construct based on the pHD-DsRed-attP plasmid.…”

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

“…However, frameshift mutations, nonsense mutations, or small deletions caused by NHEJ are unlikely to disrupt the functions of lncRNAs. Therefore, based on a previous study by Gratz et al (2014), we developed a three-component Cas9 microinjection system consisting of the Cas9 mRNA, a gene-specific gRNA, and a homologous recombination (HR) donor plasmid, simplifying the generation of knockout constructs; this system is applicable to deletion of almost any genomic locus. The efficiency of this system enabled us to successfully and rapidly delete 105 testis-specific Drosophila lncRNAs, 33 of which were revealed to play critical roles in the regulation of late spermatogenesis.…”

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila

“…The following primers were annealed to generate the DNA fragments for guide RNAs: ephrin I95 -1 (5 ′ -CTTCGATGTACCAAAAAAGGAAGA-3 ′ and 5 ′ -AAACTCTTCCTTTTTTGGTACATC-3 ′ ), ephrin I95 -2 (5 ′ -CTT CGGAATCAAATGATATTAATT-3 ′ and 5 ′ -AAACAATTAATA TCATTTGATTCC-3 ′ ), Eph-myc-1 (5 ′ -CTTCGACGGTAATCA TATTTTGGA-3´and 5´-AAACTCCAAAATATGATTACCG TC-3´), and Eph-myc-2 (5´CTTCGTACGTAAGGTGCGG TATTC-3 ′ and 5 ′ -AAACGAATACCGCACCTTACGTAC-3 ′ ). The 5 ′ and 3 ′ homology arms were inserted into the NotI and SpeI sites of the pHD-DsRed-attP (a kind gift from S. Kondo) (Gratz et al 2014), respectively, by In-Fusion (Clonetech). For the Eph-myc, the 7xMyc sequences were also inserted in addition to the 5 ′ homology arm by three-fragment In-Fusion.…”