Frequently asked questions

Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Filter using one or more categories to focus on specific topics, or use the search bar to perform a text search.

How do I identify CRISPR editing off-target sites?

The methods used to nominate off-target sites fall into two categories:

  • Empirical methods (e.g., GUIDE-seq [1], CIRCLE-seq [2], Digenome-seq [3], DISCOVER-seq [4], or other empirical methods) use wet lab experiments to identify off-target sites.
  • In silico tools (e.g., IDT CRISPR-Cas9 guide RNA design checker) attempt to identify off-target, double-stranded breaks that may occur in the genome.

Of note, there are significant differences between the off-target sites that are nominated using biochemical (in vitro), cell-based, or in vivo methods.

  1. Tsai SQ, Zheng Z, et al. (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33(2):187–197.
  2. Tsai SQ, Nguyen NT, et al. (2017) CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods 14(6):607–614.
  3. Kim D, Bae S, et al. (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12(3):237–243.
  4. Wienert B, Wyman SK, et al. (2019) Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science 364(6437):286–289.


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