The approach to the preparation of cyclic photocleavable RNA for photoactivatable CRISPR/Cas9 System

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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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E. Ivanskaya

Intitute of Chemical Biology and Funamental Medicine SB RAS; Novosibirsk State University

Email: danov@niboch.nsc.ru
Rússia, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090; ul. Pirogova 1, Novosibirsk, 630090

M. Meschaninova

Intitute of Chemical Biology and Funamental Medicine SB RAS

Email: danov@niboch.nsc.ru
Rússia, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090

M. Vorobyeva

Intitute of Chemical Biology and Funamental Medicine SB RAS

Email: danov@niboch.nsc.ru
Rússia, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090

D. Zharkov

Intitute of Chemical Biology and Funamental Medicine SB RAS; Novosibirsk State University

Email: danov@niboch.nsc.ru
Rússia, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090; ul. Pirogova 1, Novosibirsk, 630090

D. Novopashina

Intitute of Chemical Biology and Funamental Medicine SB RAS; Novosibirsk State University

Autor responsável pela correspondência
Email: danov@niboch.nsc.ru
Rússia, prosp. Acad. Lavrentjeva 8, Novosibirsk, 630090; ul. Pirogova 1, Novosibirsk, 630090

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2. Fig. 1. The proposed strategy for the functioning of the photoregulated CRISPR/Cas9 system using cyclic photoblocked crRNAs. PAM is a protospacer adjacent motif.

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3. Fig. 2. The most probable secondary structures of cyclic crRNAs: C-42-P1, C-46-P1, C-46-P2, C-48-P1 and C-48-P2, obtained using the OligoAnalyzer program (https://www.idtdna.com/calc/analyzer).

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4. Fig. 3. RP-HPLC profile of the isolated cyclization products corresponding to the initial linear oligonucleotide (blue) and cyclic crRNA (gray). Chromatography was performed in a concentration gradient of 0–50% CH3CN in 0.02 M triethylammonium acetate (pH 7.0). The peak release time of the cyclic product C-46-P1-cyc was 571 s, and that of the initial linear 5′,3′-modified oligoribonucleotide C-46-P1 was 539 s.

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5. Fig. 4. Cleavage of cyclic RNAs C-46-P1 and C-46-P2 under UV irradiation. Electrophoretic analysis in 15% denaturing PAGE: 1 – cyclic C-46-P1, 2 – UV-irradiated cyclic C-46-P1, 3 – cyclic C-46-P2, 4 – UV-irradiated cyclic C-46-P2. Irradiation wavelength – 365 nm, irradiation time – 30 min. Visualization of RNA in the gel was performed by staining with a solution of Stains-all dye. BP – bromophenol blue.

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6. Fig. 5. (a) Schematic representation of the active CRISPR/Cas9 complex; (b) electrophoretic analysis of plasmid substrate cleavage. M – set of dsDNA length markers, K– – control containing plasmid without enzyme, K+ – plasmid after cleavage with Cas9 nuclease with unmodified crRNA. UV: the “–” sign means no irradiation, the “+” sign means irradiation with UV light for 30 min at 365 nm.

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7. Fig. 6. Efficiency of plasmid DNA cleavage using irradiated and non-irradiated cyclic photocleavable crRNAs and their non-cleavable analogs. K– – intact plasmid; K+ – plasmid cleavage by Cas9 nuclease in the presence of a pair of unmodified crRNA/tracrRNA guide RNAs; UV: the “–” sign means no irradiation, the “+” sign means irradiation with UV light for 30 min at 365 nm.

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8. Scheme 1. Introduction of an azido group at the 5'-end of a 3'-alkyne-modified oligoribonucleotide.

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9. Scheme 2. Obtaining an oligoribonucleotide containing an amino group at the 3' end and a cystamine residue at the 5' end.

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10. Scheme 3. Preparation of an oligoribonucleotide containing a thiol group at the 3' end and a maleimide group at the 5' end.

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11. Scheme 4. Synthesis of cyclic oligoribonucleotide by the azide-alkyne cycloaddition method.

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12. Scheme 5. Synthesis of cyclic oligoribonucleotide by thiol-maleimide condensation.

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