Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells

By Ophir Shalem, Neville E. Sanjana, E. Hartenian, Xi Shi, David A. Scott, Tarjei S. Mikkelsen, D. Heckl, Benjamin L. Ebert, D. Root, John G Doench and 1 more. Science, 2014

4,996 citations recorded by the source corpus.

Improving Whole-Genome Screens Improved methods are needed for the knockout of individual genes in genome-scale functional screens. Wang et al. (p. 80, published online 12 December) and Shalem et al. (p. 84, published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods. Genome-editing technology allows improved positive or negative selection screens. The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/661d49dddb7470111192c80d7e4ba1a2594c3266

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Citations in the corpus

Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR (2015, methodology): “Indeed, genes whose knockout leads to strong positive selection in PLX-treated cells are in cluster 4, including NF1, NF2, MED12, CUL3 [2].”
CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling (2014, background): “…successes using Cas9 and large-scale sgRNA libraries for genetic screening (Findlay et al., 2014; Koike-Yusa et al., 2014; Sanjana et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014), the Cas9 mouse may be directly combined with genome-wide or targeted sgRNA library to…”
The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “The simplicity and accessibility of the CRISPR/Cas9 technology platform provides many advantages over other genome editing methods, and this means that loss-of-function screening is now feasible and affordable on a genomic scale (Shalem et al., 2014).”
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, background): “In recent years, viral vectors have been extensively applied to deliver CRISPR cassettes in vitro and in vivo largely owing to their high efficiency in gene delivery and long-term stable transgene expression [34, 58, 65].”
Improved vectors and genome-wide libraries for CRISPR screening (2014, methodology): “pyogenes Cas9 sgRNA sequences of the form (N)20NGG in all constitutive exons (from the procedure in (1) above) were selected as candidate targets.”
Large‐scale image‐based screening and profiling of cellular phenotypes (2017, background): “Genome-wide siRNA or CRISPR-Cas9 libraries for human genes have been developed (48,49).”
Development and Applications of CRISPR-Cas9 for Genome Engineering (2014, neutral): “selection screens in human cells (Wang et al., 2014; Shalem et al., 2014) by introducing loss-of-function mutations into early, constitutive coding exons of a different gene in each cell (Figure 6F).”