Abstract
Purpose :
Delivery of CRISPRi components in vivo with FDA-approved adeno-associated viruses (AAVs) is limited to a significantly smaller packaging size than experimental vectors such as lentivirus. We modified the smallest known, naturally occurring Cas protein Casphi (a.k.a.CasJ) to create a CRISPRi construct that allows the inclusion of larger promoters such as CMV and two gRNAs that can all be packaged into a single AAV. Our goal is to identify a combination of catalytically inactive Casphi (dCasphi) and effector protein that inhibits hypoxia-mediated induction of VEGF but does not affect basal levels.
Methods :
Cellular trafficking of dCasphi constructs was examined to identify the combinations of peptide linkers, effector proteins, and a nuclear localization signal that provided delivery to the nucleus. Effector domains from several Krüppel (“KRAB”) family members were compared to identify functional fusion proteins. Mouse liver cell lines were generated with Sleeping Beauty transposons. Functional assays included ELISA for secreted protein and qPCR for expression levels.
Results :
dCasphi-ZIM3 was the most efficacious CRISPRi construct against basal levels of VEGF mRNA from mouse liver cell lines. dCasphi-ZIM3 also attenuated basal transcription of ANGPTL4.
Conclusions :
dCasphi-ZIM3 is appropriate for blocking basal transcription, but it appears to less effective against hypoxic induction. Thus, we are currently evaluating additional effector proteins that selectively perturb hypoxic responses. The CRISPRi dCasphi-ZIM3 toolbox includes Sleeping Beauty transposons with either Dox-inducible or constitutive EF-1a promoters as well as an AAV plasmid, all with the U6 promoter and gRNA cloning sites.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.