Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors
Year: 2022
Type: article
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.
Source: eLife
DOI: 10.7554/elife.81856
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
Institutions: Howard Hughes Medical Institute, University of California, San Francisco, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Harvard University, National Institutes of Health, National Institute on Aging, National Institute of Neurological Disorders and Stroke, UCSF Helen Diller Family Comprehensive Cancer Center, Arc Research Institute, Koch Institute for Integrative Cancer Research At MIT
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
Funders: Howard Hughes Medical Institute, Pew Charitable Trusts, Charles H. Hood Foundation, Chan Zuckerberg Initiative, Maze Therapeutics, Human Frontier Science Program, National Institutes of Health, Defense Advanced Research Projects Agency, University of California, San Francisco, Ludwig Center for Molecular Oncology, Ludwig Center at Harvard, National Institute on Aging, National Human Genome Research Institute, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Institute of Neurological Disorders and Stroke, School of Medicine, University of California, San Francisco
Awards: G1473399777, The role of TMA7 in mitochondrial dysfunction, G2515541506, A GENETIC INTERACTION MAP OF THE HUMAN NUCLEUS, G3822381711, G4785822992, G5052434461, Illumina NovaSeq 6000 Sequencing System, G7436065731, G7652223712, Elucidating cellular pathways controlling the reactive state of astrocytes in Alzheimer's Disease, G9000067977, G9000102670, G9000126658, G9000255851
OpenAlex ID: W4313232103