Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

• DOI: 10.1101/2022.07.13.499814
• Publication Date: 2022-07-14T07:20:12Z
• Authors: Joseph M. Replogle; Jessica L. Bonnar; Angela N. Pogson; Christina R. Liem; Nolan K. Maier; Yufang Ding; Baylee J. Russell; Xingren Wang; Kun Leng; Alina Guna; Thomas M. Norman; Ryan A. Pak; Daniel M. Ramos; Michael E. Ward; Luke A. Gilbert; Martin Kampmann; Jonathan S. Weissman; Marco Jost
• Keywords: Subgenomic mRNA; CRISPR interference

Abstract CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions cell biology, genetics, biotechnology, but wider deployment CRISPRi screening has been constrained by large size single guide RNA (sgRNA) libraries challenges generating models with consistent knockdown. Here, we present next-generation sgRNA effector constructs that enable strong knockdown across models. First, combine empirical selection a dual-sgRNA library design generate an ultra-compact (1-3 elements per gene), highly active library. Next, rigorously compare effectors show recently published Zim3-dCas9 provides best balance between on-target minimal nonspecific effects on growth or transcriptome. Finally, engineer suite lines stable robust Our results publicly available reagents establish practices for screening.