UMMS Affiliation

RNA Therapeutics Institute; Department of Molecular, Cell, and Cancer Biology; Department of Biochemistry and Molecular Pharmacology; Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Publication Date

2016-10-13

Document Type

Article

Disciplines

Bioinformatics | Computational Biology | Genomics

Abstract

Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy.

Keywords

Cas9, coconversion, genetic screen, homologous recombination, microhomology-mediated end joining

DOI of Published Version

10.1534/g3.116.032557

Source

G3 (Bethesda). 2016 Oct 13;6(10):3197-3206. doi: 10.1534/g3.116.032557. Link to article on publisher's site

Journal/Book/Conference Title

G3 (Bethesda, Md.)

Comments

First author Daniel Tianfang Ge is a doctoral student in the Interdisciplinary Graduate Program in the Graduate School of Biomedical Sciences (GSBS) at UMass Medical School.

Related Resources

Link to Article in PubMed

PubMed ID

27543296

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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