Genetic Screens in Human Cells Using the CRISPR-Cas9 System

By Tim Wang, Jenny J. Wei, David M. Sabatini, Eric S. Lander. Science, 2013

2,804 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 bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/57fa31aa22aad401372d416dd7573c279ca20f48

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Citations in the corpus

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells (2014, neutral): “When 0.02 μg plasmid was transfected, Nanog-sg3 RNA was barely detected (lane 14); the 122 peaks identified in this library showed low overlap (9”
An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis (2015, methodology): “Experimental Procedures CRISPR-based screen The metabolism-focused sgRNA library was designed and performed as previously described (Wang et al., 2014).”
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation (2014, background): “A series of elegant studies recently exploited the readily programmable nature of Cas9, in which the specificity is determined by a short guide (sg)RNA, to enable genome-scale loss-of-function screens (Koike-Yusa et al., 2014; Shalem et al., 2014;Wang et al., 2014).”
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing (2014, background): “In addition, for CRISPR/Cas9 mode, the results are ranked by: (iii) GC-content, and (iv) the presence of a guanine at position 20 in the sgRNA target site (14,15).”
Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease (2014, methodology): “This was done using a PYTHON script with the following algorithm: 1) For every read, example (displaying columns relevant for analysis) CHROMOSOME START END CIGAR STRING chr1 526162 526361 11M1I171M1D16M [D: Deletion, I: Insertion, M: Alignment match, Numbers (11,2,24,1,12): Length of sequence with ”
CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling (2014, background): “…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 uncover novel biology.”
Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens (2017, background): “Brown [2], Matej Usaj [2], Maximilian Billmann [6], Mahfuzur Rahman [6], Michael Constanzo [2], Chad L.”