Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

By Luke A. Gilbert, Max A. Horlbeck, Britt Adamson, Jacqueline E. Villalta, Yuwen Chen, E. Whitehead, Carla P. Guimarães, B. Panning, Hidde L. Ploegh, Michael C. Bassik and 3 more. Cell, 2014

2,566 citations recorded by the source corpus.

SUMMARY While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ~1000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retro-translocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/9e2363403a9e067fca8ddb5d269ed9fcb8cb3371

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Citations in the corpus

Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 (2016, positive): “We next examined curated datasets from screens that used CRISPRa/i technology 36,40 .”
Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR (2015, background): “CRISPR screens can be performed by either direct knockout of genes using CRISPR/Cas9 [1, 2], or perturbing gene expressions using CRISPR and a dead-Cas9 (dCas9) fused with activation or repression effectors [5, 6].”
Exploring genetic interaction manifolds constructed from rich single-cell phenotypes (2019, methodology): “K562 cells stably expressing the SunTag CRISPRa sys-tem ( 11 ) were transduced with the CRISPRa GI library, and sgRNA pair abundance was compared at the start of the screen and after ten days of growth to measure fitness phenotypes.”
Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation (2016, methodology): “…genes. sgRNAs were ranked by g, and considered true or false positives if they targeted essential or non-essential genes, respectively, as defined by Evers et al. (C) Volcano plots of gene phenotypes and p-values for growth screens performed with CRISPRi v1 (Gilbert et al., 2014) and hCRISPRi-v2.”
High-Content Screening for Quantitative Cell Biology. (2016, methodology): “Genome-scale CRISPRiand CRISPRa libraries, which inhibitand activategene expression respectively, offer the opportunity to modulate gene expression over an approximately 1000-fold range [6].”
Systematic mapping of functional enhancer-promoter connections with CRISPR interference (2016, methodology): “Because the efficiency of different sgRNAs for CRISPRi can vary dramatically (10), we used a sliding window approach, averaging the scores of 20 consecutive sgRNAs and assessing the false discovery rate (FDR) of this metric through comparison to negative control, non-essential regions (13) (Fig.”
Delivering CRISPR: a review of the challenges and approaches (2018, methodology): “…the pace of biological research in many areas, including identifying and understanding mechanisms of genetic diseases (Findlay et al., 2014; Gilbert et al., 2014; Zhou et al., 2014; Konermann et al., 2015), validating disease targets (Shalem et al., 2014; Wang et al., 2014), developing…”