Targeted Genome Editing in Human Cells Using CRISPR/Cas Nucleases and Truncated Guide RNAs

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

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.

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Endonucleases / metabolism*
  • Gene Targeting*
  • Genome, Human*
  • Humans
  • Molecular Sequence Data
  • Mutagenesis
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • Endonucleases