Computationally guided high-throughput engineering of an anti-CRISPR protein for precise genome editing in human cells.

The application of CRISPR-Cas systems to genome editing has revolutionized experimental biology and is an emerging gene and cell therapy modality. CRISPR-Cas systems target off-target regions within the human genome, which is a challenge that must be addressed. Phages have evolved anti-CRISPR proteins (Acrs) to evade CRISPR-Cas-based immunity. Here, we engineer an Acr (AcrIIA4) to increase the precision of CRISPR-Cas-based genome targeting. We developed an approach that leveraged (1) computational guidance, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, ∼10,000 Acr variants were tested. Variants that improved editing precision were tested in additional validation experiments that revealed robust enhancement of gene editing precision and synergy with a high-fidelity version of Cas9. This scalable high-throughput screening framework is a promising methodology to engineer Acrs to increase gene editing precision, which could be used to improve the safety of gene editing-based therapeutics.