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

By S. Tsai, Zongli Zheng, Nhu T. Nguyen, M. Liebers, V. Topkar, V. Thapar, Nicolas Wyvekens, Cyd Khayter, A. Iafrate, Long P. Le and 2 more. 2014

2,123 citations recorded by the source corpus.

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

Subfields: Molecular Biology

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “Analysis was carried out using the previously published Python code 32 .”
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 (2016, neutral): “43 Whereas Rule Sets 1 and 2 represent data-driven, quantitative models of on-target sgRNA effectiveness, a quantitative analysis of off-target interactions based on similarly large-scale data has been lacking, and the ability to predict off-target effects therefore quite limited.”
In vivo genome editing using Staphylococcus aureus Cas9 (2015, background): “However, the off-target sites identified in vitro might differ from those in vivo , which need to be further evaluated by the in vivo applications of BLESS or other unbiased techniques such as those published during the revision of this work 43,44 .”
Whole organism lineage tracing by combinatorial and cumulative genome editing (2016, methodology): “The v1 barcode was comprised of ten targets arrayed in order 591 of decreasing activity as measured with the GUIDE-seq assay performed in human cells (22), 592 starting with the target perfectly matching the sgRNA spacer sequence.”