CRISPR Guide RNA Validation In Vitro

Zebrafish. 2017 Aug;14(4):383-386. doi: 10.1089/zeb.2016.1358. Epub 2016 Nov 9.


Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.

Keywords: CRISPR; Cas9; in vitro; knock in; validation; zebrafish.

Publication types

  • Validation Study

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Endonucleases / metabolism
  • Gene Editing*
  • Gene Targeting
  • In Vitro Techniques
  • Monophenol Monooxygenase / antagonists & inhibitors
  • Monophenol Monooxygenase / genetics
  • RNA, Guide, Kinetoplastida / genetics*
  • Zebrafish / genetics*


  • RNA, Guide
  • Monophenol Monooxygenase
  • Endonucleases