1. A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs
- Author
-
Clarence Mills, Andrew Riching, Ashleigh Keller, Jesse Stombaugh, Amanda Haupt, Elena Maksimova, Sarah M. Dickerson, Emily Anderson, Kevin Hemphill, Chris Ebmeier, John A. Schiel, Josien Levenga, Matthew Perkett, Anja van Brabant Smith, and Zaklina Strezoska
- Subjects
Genetics ,Biotechnology - Abstract
While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with
- Published
- 2022