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

By S. Gratz, Fiona P Ukken, C. D. Rubinstein, Gene H Thiede, Laura K Donohue, Alexander M. Cummings, O. KateM., Connor-Giles. Genetics, 2014

897 citations recorded by the source corpus.

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.

Subfields: Aging, Insect Science, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/612f2741291f0b78c3f10edd860ccea7948d6e16

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Citations in the corpus

Target specificity of the CRISPR-Cas9 system (2014, neutral): “The type of off-targets detected by each tool also varies; most tools only search for off-targets with few (typically three or less) PAM-proximal or total mismatches to the guide [71,75,76,79,80].”
Modelling learning and memory in Drosophila to understand Intellectual Disabilities. (2020, methodology): “The recent development of various CRISPR-based approaches for the fly (MIMIC, CRIMIC, Scarless Editing) (Venken et al., 2011a; Gratz et al., 2014, 2015a; , 2015b; Lamb et al., 2017; Li-Kroeger et al., 2018) allows researchers to model both dosagedependent mutations (loss and gain of function) more…”
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing (2014, methodology): “CHOPCHOP has a number of features that separate it from the other CRISPR/Cas9 or TALEN tools currently available (Supplementary Table S1) (25,28-34).”
Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila (2014, background): “The ubiquitous activity of vasa-cas9 presumably was masked in a previous study in which a U6:2-gRNA plasmid targeting the eye pigmentation gene rosy was injected at the posterior of the embryo, thereby restricting its activity in the vicinity of the future germ cells (11).”