TY - JOUR

T1 - Photoactivatable nanocrispr/cas9 system based on crrna reversibly immobilized on carbon nanoparticles

AU - Semikolenova, Olga

AU - Sakovina, Lubov

AU - Akhmetova, Elizaveta

AU - Kim, Daria

AU - Vokhtantsev, Ivan

AU - Golyshev, Victor

AU - Vorobyeva, Mariya

AU - Novopashin, Sergey

AU - Novopashina, Darya

N1 - Funding Information: Funding: The reported study was funded by RFBR, project number 19-34-51026, and the Russian State funded budget project of ICBFM SB RAS N 121031300042-1. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2021/10/1

Y1 - 2021/10/1

N2 - Here, we proposed a new approach to engineering a photoactivatable CRISPR/Cas9 gene-editing system. The novel nanoCRISPR/Cas9 system is based on the use of auxiliary photocleavable oligodeoxyribonucleotides (PC-DNAs) complementary to crRNA. PC-DNAs contained up to three UV-sensitive linkers made of 1-(2-nitrophenyl)-1,2-ethanediol inside the oligonucleotide chain. Immobilizing PC-DNAs on the surface of carbon nanoparticles through 3′-terminal pyrene residue provided sufficient blocking of crRNA (and corresponding Cas9 activity) before UV irradiation and allows for crRNA release after UV irradiation at 365 nm, which restores Cas9 activity. We optimized the length of blocking photocleavable oligonucleotide, number of linkers, time of irradiation, and the type of carbon nanoparticles. Based on the results, we consider the nanoCRISPR/Cas9 system involving carbon-encapsulated iron nanoparticles the most promising. It provides the greatest difference of functional activity before/after irradiation and can be used in prospective for magnetic field-controlled delivery of CRISPR system into the target cells or tissues and spatiotemporal gene editing induced by UV irradiation.

AB - Here, we proposed a new approach to engineering a photoactivatable CRISPR/Cas9 gene-editing system. The novel nanoCRISPR/Cas9 system is based on the use of auxiliary photocleavable oligodeoxyribonucleotides (PC-DNAs) complementary to crRNA. PC-DNAs contained up to three UV-sensitive linkers made of 1-(2-nitrophenyl)-1,2-ethanediol inside the oligonucleotide chain. Immobilizing PC-DNAs on the surface of carbon nanoparticles through 3′-terminal pyrene residue provided sufficient blocking of crRNA (and corresponding Cas9 activity) before UV irradiation and allows for crRNA release after UV irradiation at 365 nm, which restores Cas9 activity. We optimized the length of blocking photocleavable oligonucleotide, number of linkers, time of irradiation, and the type of carbon nanoparticles. Based on the results, we consider the nanoCRISPR/Cas9 system involving carbon-encapsulated iron nanoparticles the most promising. It provides the greatest difference of functional activity before/after irradiation and can be used in prospective for magnetic field-controlled delivery of CRISPR system into the target cells or tissues and spatiotemporal gene editing induced by UV irradiation.

KW - Carbon nanoparti-cles

KW - CRISPR/Cas9 gene editing system

KW - CrRNA immobilization

KW - Magnetic nanoparticles

KW - Photoactivation

KW - Photocleavable oligonucleotides

KW - Pyrene

UR - http://www.scopus.com/inward/record.url?scp=85116735112&partnerID=8YFLogxK

U2 - 10.3390/ijms222010919

DO - 10.3390/ijms222010919

M3 - Article

C2 - 34681578

AN - SCOPUS:85116735112

VL - 22

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 20

M1 - 10919

ER -