Research output: Contribution to journal › Article › peer-review
Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System. / Sakovina, Lubov; Vokhtantsev, Ivan; Akhmetova, Elizaveta et al.
In: International Journal of Molecular Sciences, Vol. 25, No. 22, 12392, 19.11.2024.Research output: Contribution to journal › Article › peer-review
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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 -
ID: 61148780