Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Zexiang Chen¹, Gitali Devi¹, Amena Arif¹

¹RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, United States.

ACS Chemical Biology

|April 21, 2022

Abstract:

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.

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