TY - JOUR

T1 - Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System

AU - Sakovina, Lubov

AU - Vokhtantsev, Ivan

AU - Akhmetova, Elizaveta

AU - Vorobyeva, Mariya

AU - Vorobjev, Pavel

AU - Zharkov, Dmitry O

AU - Novopashina, Darya

N1 - The reported study was funded by RSF, project number 22-14-00294, and the Russian state-funded project for ICBFM SB RAS (grant number 121031300042-1). D.O.Z. acknowledges partial salary support from the Russian Ministry of Science and Higher Education (project FSUS-2020-0035).

PY - 2024/11/19

Y1 - 2024/11/19

N2 - The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min-1 and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.

AB - The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min-1 and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.

KW - CRISPR-Cas Systems

KW - RNA, Guide, CRISPR-Cas Systems/genetics

KW - Gene Editing/methods

KW - CRISPR-Associated Protein 9/metabolism

KW - Humans

KW - Ultraviolet Rays

KW - Photolysis

KW - Light

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85210227307&origin=inward&txGid=38d93b7d73763cd910df632040b8c244

U2 - 10.3390/ijms252212392

DO - 10.3390/ijms252212392

M3 - Article

C2 - 39596457

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 22

M1 - 12392

ER -