Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple genomic loci targeting by processing numerous crRNAs from a single transcript, however, their low efficiency has hindered applications in vivo. Through structure-guided protein engineering, we develop a hyper-efficient LbCas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low crRNA conditions. We demonstrate that hyperdCas12a has minimal off-target effects compared to the wildtype system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a-activator and a single crRNA array simultaneously activating endogenous Oct4, Sox2, and Klf4 genes in the retina of postnatal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene modulation and gene therapy applications.