Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors
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 in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1–3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Dec 28, 2022
- Source ID
- 10.7554/elife.81856
Entities
People
- Alina Guna
- Angela N. Pogson
- Baylee J. Russell
- Christina R. Liem
- Daniel M. Ramos
- Jessica L. Bonnar
- Jonathan Weissman
- Joseph M Replogle
- Kun Leng
- Luke A Gilbert
- Marco Jost
- Martin Kampmann
- Michael E Ward
- Nolan K Maier
- Ryan A Pak
- Thomas M Norman
- Xingren Wang
- Yufang Ding
Organizations
- Chan Zuckerberg Initiative
- Defense Advanced Research Projects Agency
- Harvard University
- Health Resources in Action
- Howard Hughes Medical Institute
- Human Frontier Science Program
- Massachusetts Institute of Technology
- National Institute of Neurological Disorders and Stroke
- National Institute on Aging
- National Institutes of Health
- The Pew Charitable Trusts
- University of California, San Francisco
- University of California, San Francisco School of Medicine
- Whitehead Institute