Multiplex CRISPR genome regulation in the retina

Abstract

Purpose : AAV gene therapy in the age of CRISPR-based gene targeting has emerged as a potentially disruptive technology for previously incurable ocular diseases, but its applications are so far limited to monogenic diseases. The capability to perform multiplexed targeting of endogenous genes would open the field of ocular gene therapy to new applications, including polygenic diseases. Recent discovery of alternative CRISPR systems beyond the widely used SpCas9 has expanded the toolkit for genetic manipulation. CRISPR-Cas12a systems enable simultaneous targeting of multiple genomic loci by processing numerous crRNAs from a single transcript, but this capability had not yet been demonstrated in vivo, in part due to relatively low efficiency of Cas12a systems.

Methods : We used structure-guided protein engineering to develop an improved LbCas12a variant, and we tested its in vivo function through AAV-based delivery to retinal ganglion cells as well as subretinal delivery by electroporation in postnatal mice.

Results : Compared to wildtype Cas12a, our improved dCas12a variant has significantly greater efficacy in gene activation, especially at low CRISPR-RNA (crRNA) conditions. It also achieves improved gene repression and gene editing. Additionally, it has similar off-targeting effects compared to its wildtype equivalent. This new Cas12a variant achieved improved AAV-based gene editing in retinal ganglion cells in vivo. Additionally, delivery of the improved dCas12a-activator with a single crRNA array simultaneously activated multiple endogenous targets and directed the differentiation of retinal progenitor cells in retina of postnatal mice.

Conclusions : Our system enables simultaneous modulation at multiple genomic loci, thus paving the way for CRISPR-based modulation of multiple pathways or synergistic targets, such as in the case of non-monogenic diseases which consist of a large proportion of human diseases.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.