SRP485708 PRJNA1068564 GSE254101 A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and – for AcrIIA5 – indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization. Overall design: To determine the activity of AcrIIA4 and -5 mutants, E. coli libraries expressing either AcrIIA4 or -5 single point mutants were FACS sorted into 4 fractions according to Acr activity (1st fraction corresponds to low Acr activity, 4th fraction corresponds to high Acr activity). FACS-sorted E. coli cell fractions were then lysed and DNA extracted for subsequent first-stage PCR amplification of the AcrIIA4 or AcrIIA5 genes using primers with Nextera XT index overhangs. After a second stage barcoding PCR using the TG Nextera XT Index Kit v2 Set A (Illumina), the libraries were sequenced on the Illumina MiSeq system using 2x250 paired-end sequencing reagents (MiSeq Reagent Kit v2, 500 cycles). GSE254101