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Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

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High-throughput functional genomics using CRISPR-Cas9

Ophir Shalem¹, Neville E Sanjana¹, Feng Zhang¹

¹Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA; McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, and Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Nature Reviews. Genetics

|April 10, 2015

Abstract:

Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.

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