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IJMS, Vol. 25, Pages 12392: Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System
International Journal of Molecular Sciences doi: 10.3390/ijms252212392
Authors: Lubov Sakovina Ivan Vokhtantsev Elizaveta Akhmetova Mariya Vorobyeva Pavel Vorobjev Dmitry O. Zharkov Darya Novopashina
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.
