Medical Scientist Training Program, University of California, San Francisco, San Francisco, United States; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States; Whitehead Institute for Biomedical Research, Cambridge, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States; Whitehead Institute for Biomedical Research, Cambridge, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States; Whitehead Institute for Biomedical Research, Cambridge, United States
Department of Microbiology, Harvard Medical School, Boston, United States
Xingren Wang
Department of Microbiology, Harvard Medical School, Boston, United States
Kun Leng
Medical Scientist Training Program, University of California, San Francisco, San Francisco, United States; Institute for Neurodegenerative Disease, University of California, San Francisco, San Francisco, United States
Alina Guna
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Whitehead Institute for Biomedical Research, Cambridge, United States
Thomas M Norman
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
Ryan A Pak
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
Daniel M Ramos
Center for Alzheimer's Disease and Related Dementias, National Institutes of Health, Bethesda, United States; National Institute on Aging, National Institutes of Health, Bethesda, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Department of Urology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States; Arc Institute, Palo Alto, United States
Institute for Neurodegenerative Disease, University of California, San Francisco, San Francisco, United States; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States; Whitehead Institute for Biomedical Research, Cambridge, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; Department of Microbiology, Harvard Medical School, Boston, United States
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.
