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CRISPR-Cas nucleases and their nuclease-deactivated dCas variants have revolutionized the field of genome editing and gene regulation. Cas12a possesses intrinsic RNAse activity and can process multiple functional crRNAs from a single long transcript, making it a powerful tool for multiplex gene targeting. We engineered a dCas12a variant termed hyperCas12a with superior efficacy in gene editing and multiplex gene regulation, especially at restrictive crRNA concentrations. Here, we describe a step-by-step protocol for constructing and validating a crRNA array, and using it with the hyperdCas12a system for multiplex gene regulation in vivo by subretinal delivery in mice.