Open AccessA Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs
Author(s) -
Clarence Mills,
Andrew S. Riching,
A.N. Keller,
Jesse Stombaugh,
Amanda Haupt,
Elena Maksimova,
Sarah Dickerson,
Emily M. Anderson,
Kevin Hemphill,
Chris Ebmeier,
John A. Schiel,
Josien Levenga,
Matthew R. Perkett,
Anja van Brabant Smith,
Žaklina Strezoska
Publication year - 2022
Publication title -
the crispr journal
Language(s) - English
Resource type - Journals
eISSN - 2573-1602
pISSN - 2573-1599
DOI - 10.1089/crispr.2022.0056
Subject(s) - crispr , cas9 , effector , guide rna , biology , rna interference , computational biology , crispr interference , gene , hek 293 cells , rna , genetics , microbiology and biotechnology
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 in vitro -transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.