An engineered, compact Cas12m variant for multiplexed gene activation in vivo.

Abstract

Achieving coordinated CRISPR-based transactivation (CRISPRa) regulation in vivo remains challenging, as the requisite genetic elements frequently exceed the packaging capacity of AAV vectors. Here we report the identification and engineering of the Cas12m protein family, a naturally nuclease-dead CRISPR system that possesses intrinsic self-processing activity for its precursor crRNA. We develop an optimized variant, denCas12m, and leverage it in the smCRISPRa platform. We show that a single transcriptional unit encoding a multi-gRNA array can be processed into individual functional guides within a target cell, enabling efficient synergistic activation of a single gene through multiple sites or simultaneous activation of distinct endogenous genes from a single all-in-one AAV vector. We demonstrate phenotypic rescue in mouse models of Duchenne muscular dystrophy and Scn1a haploinsufficiency. Furthermore, a single all-in-one AAV vector supports the simultaneous activation of multiple distinct endogenous genes in mice, with a single vector delivering up to eight crRNAs for activation of six different genes, demonstrating the platform's high-order multiplexing.