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Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.

Authors
  • 1
  • 2
  • 3
  • 1 Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
  • 2 Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
  • 3 Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA. Electronic address: [email protected]
Type
Published Article
Journal
Methods in enzymology
1557-7988
Publication Date
Volume
546
Pages
47–78
Identifiers
DOI: 10.1016/B978-0-12-801185-0.00003-9
PMID: 25398335
Source
Medline
Keywords
License
Unknown

Abstract

The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this chapter, we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~10(12)-membered) libraries of potential nuclease substrates.

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