Abstract
Standard approaches for dCas9-based modification of gene expression are limited in the ability to multiplex targets, establish streamlined cassettes, and utilize commonly studied Pol II promoters. In this work, we repurpose the dCas9-VPR activator to act as a dual-mode activator/repressor that can be programmed solely on the basis of target position at gene loci. Furthermore, we implement this approach using a streamlined Pol II-ribozyme system that allows expression of many sgRNAs from a single transcript. By "stepping" dCas9-VPR within the promoter region and ORF we create graded activation and repression (respectively) of target genes, allowing precise control over multiplexed gene modulation. Expression from the Pol II system increased the net amount of sgRNA production in cells by 3.88-fold relative to the Pol III SNR52 promoter, leading to a significant improvement in dCas9-VPR repression strength. Finally, we utilize our Pol II system to create galactose-inducible switching of gene expression states and multiplex constructs capable of modulating up to 4 native genes from a single vector. Our approach represents a significant step toward minimizing DNA required to assemble CRISPR systems in eukaryotes while enhancing the efficacy (greater repression strength), scale (more sgRNAs), and scope (inducibility) of dCas9-mediated gene regulation.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.