Format

Send to

Choose Destination
Science. 2014 Jan 3;343(6166):80-4. doi: 10.1126/science.1246981. Epub 2013 Dec 12.

Genetic screens in human cells using the CRISPR-Cas9 system.

Author information

1
Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.

Abstract

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.

PMID:
24336569
PMCID:
PMC3972032
DOI:
10.1126/science.1246981
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center