An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination

Abstract

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

The effect of repeat length on Marcal1-dependent single-strand annealing in Drosophila

“… Kanca et al (2022) showed that SSA can be used for CRISPR/Cas9 genome engineering in Drosophila . Although these authors achieved success with only 200 bp of homology on each side of the DSB our work suggests that increasing this will enhance the success of integration, yielding more efficient gene edits per round of embryonic injections.…”

“…To gain insight into how Marcal1 promotes SSA through its annealing activity, we asked how changing the length of available complementarity would affect SSA efficiency in both wild-type and Marcal1 mutant flies. This question is also of interest because SSA can be used for CRISPR/Cas9-based genome editing to incorporate fragments into the Drosophila genome ( Kanca et al 2022 ). Demonstration of this approach employed a plasmid with 200 nt of flanking homology to either side of 2 Cas9 genomic targets.…”

Drosophila melanogaster as a versatile model organism to study genetic epilepsies: An overview

“…The lower rescuing capacity of patient variants corroborated a LOF effect. As not all genes are suitable targets for CRMIC-based integration of a trojan exon, the gene can also be replaced completely by a GAL4-expressing sequence ( Kanca et al, 2022 ). Large libraries of flies with integration sites in different genes provide broad access to the Drosophila genome ( Venken et al, 2011 ; Nagarkar-Jaiswal et al, 2015 ; Lee et al, 2018 ).…”

Promoting validation and cross-phylogenetic integration in model organism research

“…CRISPR can also be used to generate mutations that are allelic orthologs to specific human disease mutations, to functionally inhibit gene/protein function in particular tissues for assessment of cell- and tissue-specific phenotypes, and to assess expression patterns and subcellular protein localization. CRISPR technology has been used to create multiple disease models by insertion of the yeast GAL4 gene in flies ( Lee et al, 2018 ; Kanca et al, 2019 , 202 1), worms ( Wang et al, 2017a ) and, to some extent, mice ( Clark et al, 2020 ). Embryonic lethal mutant phenotypes may be pursued using cell- or tissue-specific knockouts, which are more easily applied to invertebrates and zebrafish, the externally growing embryos of which allow the facile study of mutant phenotypes at cell resolution prior to death.…”