Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas. 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. 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. 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. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. 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. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. 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. 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. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. ![]() Our tools are designed for use in Drosophila but the lessons we draw are likely to be widely relevant. ![]() Importantly, we also highlight unanticipated failures, which serve as cautionary tales, and suggest mitigating measures. We describe how these tools and protocols can be implemented easily and efficiently. In a second class of modifications, essential sequences are deleted and replaced with an integrase site, which serves as a platform for the creation of custom reporters, transcriptional drivers, conditional alleles and regulatory mutations. In one class of applications, a single site-specific insertion generates a transcriptional reporter, a loss-of function allele, and a tagged allele. homology-based repair, site-specific integration and Cre or FLP-mediated recombination) to create sophisticated tools to measure and manipulate gene activity. Here we describe how CRISPR/Cas can be combined with other DNA manipulation techniques (e.g. This technology is now routine for many organisms and cell lines. CRISPR/Cas technology allows the creation of double strand breaks and hence loss of function mutations at any location in the genome.
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