Harnessing anti-CRISPR to suppress, subtract, and segregate Cas9 activity for precision CRISPR in Drosophila

Tissue-specific CRISPR (ts-CRISPR) is a powerful approach for studying cell and developmental biology by restricting mutagenesis to specific tissues. However, the precision of this approach is often compromised by non-specific, "leaky" Cas9 activity that confounds phenotypic analysis and destabilizes Cas9/gRNA stocks. To address these limitations in Drosophila, we developed a toolkit based on the Anti-CRISPR (Acr) protein AcrIIA4. We first identified AcrIIA4 as a potent in vivo Cas9 inhibitor with high stability and established the temporal requirements for its function. Based on these findings, we generated three classes of Acr tools. First, a collection of AcrIIA4-bearing balancers robustly suppresses Cas9 and enables the stable maintenance of complex Cas9/gRNA stocks. Importantly, maternal deposition of AcrIIA4 from these balancers provides a means of temporal control, delaying Cas9 activity until metamorphosis. Second, tissue-specific AcrIIA4 transgenes refine leaky Cas9 drivers in a "tissue-subtraction" strategy. Finally, germline-specific and soma-specific Acr tools efficiently segregate Cas9 activity, solving bidirectional leakiness between these two compartments. This comprehensive AcrIIA4 toolkit provides new levels of precision, versatility, and temporal control for Drosophila CRISPR applications.