GSBS Student Publications

Student Author(s)

Hui-Min Chen

GSBS Program

Neuroscience

UMMS Affiliation

Department of Neurobiology; Tzumin Lee Lab; Graduate School of Biomedical Sciences, Neuroscience Program

Date

7-22-2014

Document Type

Article

Medical Subject Headings

Alleles; Animals; Animals, Genetically Modified; Base Sequence; CRISPR-Cas Systems; DNA Repair; Drosophila melanogaster; Gene Targeting; Genes, Essential; Genes, Insect; *Genetic Engineering; Genetic Vectors; Genome, Insect; Germ Cells; Molecular Sequence Data; Mutation; Phenotype; RNA; Transgenes

Disciplines

Computational Biology | Genetics | Genomics | Molecular Genetics

Abstract

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

Rights and Permissions

Citation: Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76. doi: 10.1073/pnas.1405500111. Epub 2014 Jul 7. Link to article on publisher's site

DOI of Published Version

10.1073/pnas.1405500111

Comments

Freely available online through the PNAS open access option.

Related Resources

Link to Article in PubMed

Journal Title

Proceedings of the National Academy of Sciences of the United States of America

PubMed ID

25002478

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