Photocleavable Guide RNA for Photocontrolable CRISPR/Cas9 System

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Resumo

The development of gene editing systems on the base of CRISPR/Cas having higher efficacy, specificity, and possibility of their activity regulation by light irradiation is actually problem. Modification of CRISPR/Cas components, in particular guide RNA, by introduction of photocleavable linkers is the prospective approach for the solution of this problem. We developed the approach for the synthesis of photocleavable guide sgRNA for the CRISPR/Cas9 system containing the linkers on the base of 1-(2-nitrophenyl)-1,2-ethanediol. Such photomodified guide RNAs degrade upon UV-irradiation and CRISPR/Cas9 system is inactivated. We obtained three variants of photomodified sgRNA with different photolinker positions. It was demonstrated that sgRNA variant with photolinker introduced in the Cas9 protein binding and hairpins formation region is able to effectively guide Cas9 nuclease for DNA-target cleavage before UV-irradiation and lose its activity after irradiation. The conditions of controllable 40%-cleavage of model DNA-target were chosen. Developed approach provide specific inactivation of CRISPR/Cas9 gene editing system in specific time moment in definite place. Photoregulation of gene editing system permits not only reduce the undesirable off-target effects, but also becomes the basis of genetic disease therapy.

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Sobre autores

E. Akhmetova

Institute of Chemical Biology and Fundamental Medicine SB RAS

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

I. Vokhtancev

Institute of Chemical Biology and Fundamental 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

Institute of Chemical Biology and Fundamental Medicine SB RAS

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

M. Vorobyeva

Institute of Chemical Biology and Fundamental Medicine SB RAS

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

D. Zharvov

Institute of Chemical Biology and Fundamental 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

Institute of Chemical Biology and Fundamental 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. Cleavage of pBluescriptII SK(−) plasmid by the CRISPR/Cas9 system. (a) Schematic representation of the complex of target DNA and unmodified sgRNA with the Cas9 protein and the sequence of photolabile guide RNAs sgRNA-PL1, sgRNA-PL2, and sgRNA-PL3; (b) electrophoretic analysis of the cleavage products in 1% agarose gel. M is a DNA length marker, K⁻ is the original circular supercoiled plasmid target DNA containing a small amount of DNA in the relaxed form; K⁺ are the products of target DNA cleavage in the presence of unmodified chemically synthesized 102-nt sgRNA; other lanes – analysis of the products of target DNA cleavage in the presence of irradiated (+UV) and non-irradiated (–UV) photolabile guide RNAs (sgRNA-PL1, sgRNA-PL2 or sgRNA-PL3). Reaction time 60 min, temperature 37°C. See the “Experimental section” for reaction conditions.

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3. Fig. 2. Analysis of the efficiency of cleavage of the dsDNA target by the CRISPR/Cas9 system containing photolabile guide RNAs before and after irradiation. For cleavage conditions, see the “Experimental section.” Data were obtained by averaging the results of at least three independent experiments.

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4. Fig. 3. Electrophoretic analysis of the products of pBluescriptII SK(−) plasmid cleavage in the presence of sgRNA fragments in 1% agarose gel. M is a DNA length marker, K⁻ is the original circular supercoiled plasmid target DNA containing an insignificant amount of DNA in the relaxed form; K+ is the product of target DNA cleavage in the presence of unmodified chemically synthesized sgRNA; (a) sgRNA-PL2 is the products of target DNA cleavage in the presence of 5ʹfr_sgRNA-PL2 and 3ʹfr_sgRNA-PL2 (5ʹfr +3ʹfr) or only 5ʹfr_sgRNA-PL2 (5ʹfr); (b) – sgRNA-PL3 (69) – products of target DNA cleavage in the presence of the 69-mer 5ʹ-fragment of sgRNA-PL3; (c) – analysis of the efficiency of plasmid cleavage by Cas9 nuclease in the presence of sgRNA fragments. For cleavage conditions, see the “Experimental section”. The data were obtained by averaging the results of at least two independent experiments.

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5. Fig. 4. Kinetic curves of target DNA cleavage by Cas9 complexes with unmodified guide RNAs, obtained by processing the data of electrophoretic analysis of reaction mixtures in agarose gel with visualization by ethidium bromide. For cleavage conditions, see the “Experimental section”. The data were obtained by averaging the results of at least three independent experiments.

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6. Fig. 5. Kinetic curves of target DNA cleavage by the Cas9 protein complex with sgRNA-PL2. 1 – control RNA (sgRNA), 2 – photosensitive RNA (sgRNA-PL2), 3 – deactivated photosensitive RNA in complex with Cas9, 4 – reaction stopped by UV irradiation (30 min) from the 5th minute from the start of the reaction, 5 – reaction stopped by UV irradiation (5 min) from the 5th minute from the start of the reaction, 6 – photosensitive RNA deactivated outside the complex with Cas9. For cleavage conditions, see the “Experimental section”. The data were obtained by averaging the results of at least three independent experiments.

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