Here authors 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. Authors also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair towards HDR. Finally, group developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules.
Reference: Gratz, Scott J., et al. "Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila." Genetics 196.4 (2014): 961-971.
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