Understanding GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

GUIDE-Seq is a novel method for detecting genome-wide DNA double-stranded breaks (DSBs) induced by CRISPR RNA-guided nucleases (RGNs) and other nucleases. The method involves capturing double-stranded oligodeoxynucleotides (dsODNs) into DSBs and mapping their integration sites using next-generation sequencing. This approach revealed wide variability in RGN off-target activities and identified many previously unknown off-target sites. GUIDE-Seq also detected RGN-independent genomic breakpoint hotspots and showed that truncated guide RNAs significantly reduced off-target DSBs. The method provides a rigorous framework for evaluating RGN specificity and safety before clinical use.GUIDE-Seq is a novel method for detecting genome-wide DNA double-stranded breaks (DSBs) induced by CRISPR RNA-guided nucleases (RGNs) and other nucleases. The method involves capturing double-stranded oligodeoxynucleotides (dsODNs) into DSBs and mapping their integration sites using next-generation sequencing. This approach revealed wide variability in RGN off-target activities and identified many previously unknown off-target sites. GUIDE-Seq also detected RGN-independent genomic breakpoint hotspots and showed that truncated guide RNAs significantly reduced off-target DSBs. The method provides a rigorous framework for evaluating RGN specificity and safety before clinical use.

GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

2015 February ; 33(2): 187–197. doi:10.1038/nbt.3117. | Shengdar Q. Tsai#1,2,3, Zongli Zheng#1,2,3,4, Nhu T. Nguyen1,2, Matthew Liebers1,2, Ved V. Topkar1,2, Vishal Thapar1,2, Nicolas Wyvekens1,2, Cyd Khayter1,2, A. John Iafrate1,2,3, Long P. Le1,2,3, Martin J. Aryee1,2,3, and J. Keith Joung1,2,3