Higher resolution pooled genome-wide CRISPR knockout screening in Drosophila cells using integration and anti-CRISPR (IntAC).

ABSTRACT: CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries. The method relies on weak sgRNA promoters to avoid early CRISPR-Cas9 activity causing discrepancies between genome edits and integrated sgRNAs. To address this issue and utilize higher strength sgRNA expression, we introduce a method to co-transfect a plasmid expressing anti-CRISPR protein to suppress early CRISPR-Cas9 activity which we term "IntAC" (integrase with anti-CRISPR). IntAC dramatically improves precision-recall of fitness genes across the genome, allowing us to generate the most comprehensive list of cell

SUBMITTER: Viswanatha R 

PROVIDER: S-EPMC12259957 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature