title: Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research.
abstract: The Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated (CRISPR/Cas) technology allows rapid, site-specific genome modification in a wide variety of organisms . Proof-of-principle studies in Drosophila melanogaster have used various CRISPR/Cas tools and experimental designs, leading to significant uncertainty in the community about how to put this technology into practice. Moreover, it is unclear what proportion of genomic target sites can be modified with high efficiency. Here, we address these issues by systematically evaluating available CRISPR/Cas reagents and methods in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for somatic and germline genome engineering and conclude that optimized CRISPR with independent transgenes is as efficient, more versatile, and does not represent a biosafety risk.
authors.affiliation: Cell Biology Division, MRC-Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom fport@mrc-lmb.cam.ac.uk sbullock@mrc-lmb.cam.ac.uk.
authors.forename: Fillip
authors.initials: F
authors.lastname: Port
authors.affiliation: Cell Biology Division, MRC-Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Nadine
authors.initials: N
authors.lastname: Muschalik
authors.affiliation: Cell Biology Division, MRC-Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom fport@mrc-lmb.cam.ac.uk sbullock@mrc-lmb.cam.ac.uk.
authors.forename: Simon L
authors.initials: SL
authors.lastname: Bullock
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004330:Drosophila / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D055879:Gene Knock-In Techniques; D005854:Germ Cells; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR; Drosophila; gene drive; homology-directed repair; mutagenesis