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Methods Enzymol. 2014;546:21-45. doi: 10.1016/B978-0-12-801185-0.00002-7.

Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.

Author information

1
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
2
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: jjoung@mgh.harvard.edu.

Abstract

CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.

KEYWORDS:

CRISPR RNA-guided nucleases; CRISPR/Cas nucleases; Cas9; Genome editing; Off-target effects; Off-target mutations; Truncated guide RNAs

[Indexed for MEDLINE]

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