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 pooled CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries, followed by PCR and sequencing of integrated sgRNAs. While effective, the method relies on early constitutive Cas9 activity that potentially can lead to discrepancies between genome edits and sgRNAs detected by PCR, reducing screen accuracy. To address this issue, we introduce a new method to co-transfect a plasmid expressing the anti-CRISPR protein AcrIIa4 to suppress Cas9 activity during early sgRNA expression, which we term "IntAC" (integrase with anti-CRISPR). IntAC allowed us to construct a new CRI

SUBMITTER: Viswanatha R 

PROVIDER: S-EPMC11429967 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature