The approach to the preparation of cyclic photocleavable RNA for photoactivatable CRISPR/Cas9 System
- Authors: Ivanskaya E.V.1,2, Meschaninova M.I.1, Vorobyeva M.A.1, Zharkov D.O.1,2, Novopashina D.S.1,2
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Affiliations:
- Intitute of Chemical Biology and Funamental Medicine SB RAS
- Novosibirsk State University
- Issue: Vol 50, No 5 (2024)
- Pages: 622-635
- Section: Articles
- URL: https://archivog.com/0132-3423/article/view/670795
- DOI: https://doi.org/10.31857/S0132342324050051
- EDN: https://elibrary.ru/LRJNBW
- ID: 670795
Cite item
Abstract
The development of controllable gene editing systems on the base of CRISPR/Cas is an actually problem of modern molecular biology and genetic enginery. Interesting variant of solution of this problem is modification of guide RNA by introduction of photocleavable linkers. We developed the approach to the synthesis of cyclic photocleavable guide crRNA for the CRISPR/Cas9 system with photolinker on the base of 1-(2-nitrophenyl)-1,2-ethanediol (PL). Upon irradiation by UV-light these guide RNA are linearized and CRISPR/Cas9 system is activated. Two chemical methods to the cyclization of RNA were tested: Michael reaction (thiol-maleimide condensation) and Cu-catalyzed azide-alkyne cycloaddition (CuAAC, click-chemistry reaction). For this purpose 5',3'-modified RNA containing reactive groups were prepared. The advantages of CuAAC reaction for cyclic RNA preparation was demonstrated. Effectivity of cyclic RNAs is depends from their secondary structure and ability of reactive groups to draw together. Series of photocleavable and control non-cleavable cyclic crRNA were obtained. It was shown that cyclic crRNAs guide nuclease Cas9 for plasmid cleavage less effective but linearization of photocleavable cyclic crRNA increases extent of plasmid cleavage. Developed approach permits prepare cyclic photocleavable RNA including spatiotemporal activation of guide RNA for gene editing. Photoregulation of gene editing will permit to lower the off-target effects and to carry out the editing more targeting.
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About the authors
E. V. Ivanskaya
Intitute of Chemical Biology and Funamental Medicine SB RAS; Novosibirsk State University
Email: danov@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090; ul. Pirogova 1, Novosibirsk, 630090
M. I. Meschaninova
Intitute of Chemical Biology and Funamental Medicine SB RAS
Email: danov@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090
M. A. Vorobyeva
Intitute of Chemical Biology and Funamental Medicine SB RAS
Email: danov@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090
D. O. Zharkov
Intitute of Chemical Biology and Funamental Medicine SB RAS; Novosibirsk State University
Email: danov@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090; ul. Pirogova 1, Novosibirsk, 630090
D. S. Novopashina
Intitute of Chemical Biology and Funamental Medicine SB RAS; Novosibirsk State University
Author for correspondence.
Email: danov@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090; ul. Pirogova 1, Novosibirsk, 630090
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