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

Raghuvir Viswanatha (), Samuel Entwisle, Yanhui Hu, Ah-Ram Kim, Kelly Reap, Matthew Butnaru, Mujeeb Qadiri, Stephanie E. Mohr and Norbert Perrimon ()
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Raghuvir Viswanatha: Harvard Medical School
Samuel Entwisle: Harvard Medical School
Yanhui Hu: Harvard Medical School
Ah-Ram Kim: Harvard Medical School
Kelly Reap: Harvard Medical School
Matthew Butnaru: Harvard Medical School
Mujeeb Qadiri: Harvard Medical School
Stephanie E. Mohr: Harvard Medical School
Norbert Perrimon: Harvard Medical School

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: 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 fitness genes yet assembled for Drosophila. Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We also perform a resistance screen to proaerolysin, a glycosylphosphatidylinositol-(GPI)-binding pore-forming toxin, retrieving 18/23 expected and one previously uncharacterized GPI synthesis gene. We also demonstrate that an IntAC sublibrary enables precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell and species types.

Date: 2025
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DOI: 10.1038/s41467-025-61692-3

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