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Genome Engineering Using CRISPR-Cas9 System

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Book cover Chromosomal Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1239))

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

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Author information

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA, 02142, USA

    Le Cong & Feng Zhang

  2. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Le Cong & Feng Zhang

Authors
  1. Le Cong
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  2. Feng Zhang
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Corresponding author

Correspondence to Le Cong .

Editor information

Editors and Affiliations

  1. Department of Genetics, Genome Engineering and iPSC Center, Washington University School of Medicine, St. Louis, Missouri, USA

    Shondra M. Pruett-Miller

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Cong, L., Zhang, F. (2015). Genome Engineering Using CRISPR-Cas9 System. In: Pruett-Miller, S. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 1239. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_10

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  • DOI: https://doi.org/10.1007/978-1-4939-1862-1_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1861-4

  • Online ISBN: 978-1-4939-1862-1

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