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Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing.

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.

SUBMITTER: Chen Z

PROVIDER: S-EPMC9127786 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing.

Chen Zexiang Z Devi Gitali G Arif Amena A Zamore Phillip D PD Sontheimer Erik J EJ Watts Jonathan K JK

ACS chemical biology 20220421 5

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 succes ...[more]

PMID: 35446558

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