Abstract
We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.
Highlights
A main goal of the Drosophila Gene Disruption Project (GDP) is to create genetic tools that facilitate an integrated approach to analyze the function of each gene in detail
This involves assessment of the loss of function phenotype, identification of the cells that express the gene, determination of the subcellular protein localization, selective removal of the transcript or protein in any tissue, the ability to perform immunoprecipitation of the protein and its interacting proteins or DNA, rescue of the induced fly mutant phenotypes with fly or human cDNAs and assessment of the consequences of amino acid variants in vivo. These elegant and precise manipulations are made possible by the integration of a Swappable Integration Cassette (SIC) in the gene of interest (GOI) using transposon mediated integration [Minos-mediated Integration Cassette (MiMIC) (Venken et al, 2011; Nagarkar-Jaiswal et al, 2015a)] or homologous recombination mediated by CRISPR, a technique we named CRIMIC (CRISPR-mediated Integration Cassette) (Zhang et al, 2014; Diao et al, 2015; Lee et al, 2018b)
The CRIMIC variety of SIC currently used by the GDP is an artificial exon consisting of attP-FRT-SA-T2A-GAL4polyA-3XP3-EGFP-FRT-attP inserted in a coding intron of the GOI (Lee et al, 2018b)
Summary
A main goal of the Drosophila Gene Disruption Project (GDP) is to create genetic tools that facilitate an integrated approach to analyze the function of each gene in detail This involves assessment of the loss of function phenotype, identification of the cells that express the gene, determination of the subcellular protein localization, selective removal of the transcript or protein in any tissue, the ability to perform immunoprecipitation of the protein and its interacting proteins or DNA, rescue of the induced fly mutant phenotypes with fly or human cDNAs and assessment of the consequences of amino acid variants in vivo. We anticipate that the drop-in approaches will be useful in other species
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