Fig. 1: Schematic of the IntAC system and validation of transient inhibition by anti-CRISPR in Drosophila cells. | Nature Communications

Fig. 1: Schematic of the IntAC system and validation of transient inhibition by anti-CRISPR in Drosophila cells.

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

Fig. 1: Schematic of the IntAC system and validation of transient inhibition by anti-CRISPR in Drosophila cells.

Schematic representation of the current CRISPR screening system compared with the IntAC system. A In the v.1 approach, sgRNAs are active upon pooled transfection and one of these at random integrates into the cell’s genome via φC31 integration. Following cell divisions and the loss of transiently transfected plasmids, one sgRNA, illustrated by sgA (purple), is found in the cell’s genome, but undesired cutting by other sgRNAs may have occurred, illustrated by sgB (orange). This is tolerable due to the weaker promoter for sgRNAs (U6:2). Elements created in BioRender. Viswanatha, R. (2025) https://BioRender.com/0w8vn0z. B In the v.2 approach, anti-CRISPR (red) is expressed, initially inactivating Cas9 and preventing CRISPR, while one sgRNA is still integrated into the cell’s genome using φC31 integration. Later, following cell divisions, transiently transfected sgRNAs are lost, and the integrated sgRNA alone is active in the cell. We also used the stronger U6:3 promoter in v.2 to express more sgRNA. pInt = φC31 Integrase expression plasmid. pIntAC =  φC31-T2A-AcrIIa4 expression plasmid. C T7 endonuclease I-sensitivity assay, showing the edited versus unedited allele targeted by a CRISPR sgRNA measured over time in cells transfected as indicated. Cell populations transfected with IntAC exhibited a clear early delay in editing, with editing efficiency returning to nearly that of controls by day 18. Quantification of editing (edited band intensity divided by the sum of the intensities of the edited and unedited bands). Representative of three experimental replicates. Molecular weight markers are given in kilobases. Created in BioRender. Viswanatha, R. (2025) https://BioRender.com/zmb3fwf. D Phenotypic validation of IntAC-delayed editing. Cells with Rho1 suppression, characterized by large cell morphology, were observed later in the IntAC population compared to controls, confirming the temporal delay in Cas9 activity. Red = mCherry in Drosophila S2R+ derivative PT5 (NPT005; DGRC #229) cells; Green = free GFP expression from sgRNA plasmid (pLib6.6/sgRho1) which additionally encodes Actin promoter-driven GFP.

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