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      Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

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

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

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          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.

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          Author and article information

          Journal
          10.1073/pnas.1405500111
          4115528
          25002478

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