Development of a Facile Approach for Generating Chemically Modified CRISPR/Cas9 RNA

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Scott, T
Soemardy, C
Morris, KV
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2020
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Abstract

The RNA-guided, modified type II prokaryotic CRISPR with CRISPR-associated proteins (CRISPR/Cas9) system represents a simple gene-editing platform with applications in biotechnology and also potentially as a therapeutic modality. The system requires a small guide RNA (sgRNA) and a catalytic Cas9 protein to induce non-homologous end joining (NHEJ) at break sites, resulting in the formation of inactivating mutations, or through homology-directed repair (HDR) can engineer in specific sequence changes. Although CRISPR/Cas9 is a powerful technology, the effects can be limited as a result of nuclease-mediated degradation of the RNA components. Significant research has focused on the solid-phase synthesis of CRISPR RNA components with chemically modified bases, but this approach is technically challenging and expensive. Development of a simple, generic approach to generate chemically modified CRISPR RNAs may broaden applications that require nuclease-resistant CRISPR components. We report here the development of a novel, functional U-replaced trans-activating RNA (tracrRNA) that can be in vitro transcribed with chemically stabilizing 2′-fluoro (2′F)-pyrimidines. These data represent a unique and facile approach to generating chemically stabilized CRISPR RNA.

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Molecular Therapy - Nucleic Acids

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19

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© 2020 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International (CC BY-NC-ND 4.0) License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, providing that the work is properly cited.

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Biochemistry and cell biology

Clinical sciences

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Scott, T; Soemardy, C; Morris, KV, Development of a Facile Approach for Generating Chemically Modified CRISPR/Cas9 RNA, Molecular Therapy - Nucleic Acids, 2020, 19, pp. 1176-1185

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