Alekseev, A.; Klimko, V.; Frantsuzova, I.; Abramova, M.; Vinnik, V.; Vasileva, A.; Selkova, P.; Khodorkovskii, M.; Arseniev, A.

CRISPR-Cas12d is a distinct V-D type system discovered in metagenomes of Candida Phyla Radiation bacteria. It stands out from most closely related systems due to its 17-19 nucleotide short spacer region and specialized stabilizing scout RNAs. We have made significant improvements to this system by modifying its scout RNA to create sgRNA, which greatly simplifies its use. We found mutations in the RuvC domain of the effector protein KbCas12d that result in loss of nuclease activity. We obtained two catalytically inactive dKb[C]as12d variants D827A and E913A. Using the optical tweezers technique, we demonstrated high specificity of dKb[C]as12d in binding targets on individual DNA molecules. Engineered sgRNA and catalytically inactive dKb[C]as12d variants have promising applications in biotechnology for the precise regulation of gene expression and molecular diagnostics.