Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing

ACS Chem Biol. 2022 May 20;17(5):1045-1050. doi: 10.1021/acschembio.2c00116. Epub 2022 Apr 21.


CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Gene Editing* / methods
  • Humans
  • RNA, Guide, CRISPR-Cas Systems* / chemistry
  • RNA, Guide, CRISPR-Cas Systems* / genetics


  • RNA, Guide, CRISPR-Cas Systems