Proteasomal control of anti-CRISPRs for the regulation of CRISPR-Cas9 activity using Cas9-ACROBAT.

Abstract

Precise regulation of CRISPR-Cas9 activity is critical to limit off-target effects and improve the safety of genome engineering. To achieve reversible and robust control of Cas9, we developed multiple small-molecule-responsive systems that modulate the stability of the anti-CRISPR protein AcrIIA4. By fusing AcrIIA4 to dTAG, SMASh, or ecDHFR degrons, we enabled ligand-dependent degradation or stabilization of the inhibitor, thereby tightly regulating CRISPR cutting, activation, and interference in human cells. To further eliminate background Cas9 activity, we engineered a theophylline-responsive riboswitch into the sgRNA scaffold to block Cas9-sgRNA assembly until ligand addition. Combining riboswitch sgRNAs with degron-controlled AcrIIA4 produced the Cas9-ACROBAT platform, which provided near-complete suppression of CRISPR leakiness, while maintaining potent induction of genome editing and transcriptional modulation, including regulation of endogenous targets such as PLK1, CDKN1A, and CCND1. Cas9-ACROBAT offers a versatile, modular strategy for precise, small-molecule control of CRISPR systems.

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