Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors
- University of California, San Francisco, United States
- Whitehead Institute for Biomedical Research, United States
- Harvard Medical School, United States
- National Institutes of Health, United States
- National Institute of Neurological Disorders and Stroke, United States
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 nonspecific 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.
Data availability
Sequencing data are available on NCBI GEO under accession number GSE205310 (Perturb-seq) and GSE205147 (bulk RNA-seq). sgRNA counts from CRISPRi screens are included as supplemental tables. All data generated or analyzed during this study are included in the manuscript and supporting files.
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Comparison of dual sgRNA library versus Dolcetto by Perturb-seqNCBI Gene Expression Omnibus, GSE205310.
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Comparison of non-specific transcriptional effects of CRISPRi effector proteins by RNA-seqNCBI Gene Expression Omnibus, GSE205147.
Article and author information
Author details
Michael Emmerson Ward
Funding
National Institutes of Health (R00GM130964)
- Marco Jost
National Institutes of Health (T32AI132120)
- Baylee J Russell
Human Frontier Science Program (2019L/LT000858)
- Alina Guna
Chan Zuckerberg Initiative (Ben Barres Early Career Acceleration Award)
- Martin Kampmann
Howard Hughes Medical Institute (Investigator)
- Jonathan S Weissman
National Institutes of Health (RM1HG009490-01)
- Jonathan S Weissman
Springer Nature Global Grant for Gut Health (1772808)
- Marco Jost
Charles H. Hood Foundation (Child Health Research Award)
- Marco Jost
Defense Advanced Research Projects Agency (HR0011-19-2-0007)
- Jonathan S Weissman
Ludwig Center for Molecular Oncology (NA)
- Jonathan S Weissman
Chan Zuckerberg Initiative (NA)
- Jonathan S Weissman
National Institutes of Health (F31NS115380)
- Joseph M Replogle
National Institutes of Health (F30AG066418)
- Kun Leng
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors
eLife 11:e81856.
https://doi.org/10.7554/eLife.81856
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Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.
