Part:BBa_K4778015
scgRNA-F
Biology
Sequence：
iDNA-scgRNA Handle：5’-ATC TAC ACT T/i6FAMdT/TTATT/iBHQ1dT/AGT AGA AAT TA -3’
iDNA-scgRNA Spacer：5’-TCA TAG TTA G/i6FAMdT/TTATT/iBHQ1dT/CGT AAC GAT C -3’
sgRNA：5’-UAAUUUCUACUAAGUGUAGAUGAUCGUUACGCUAACUAUGA -3’
Length: 41bp
It is the substrate of Cas12a_sgRNA_D_assistantDNA_D with ssDNA trans-cleaving activity in the 2023 project of AFMU_China team. This design comes from the CONAN system and has the ability of signal exponential amplification and fluorescence release. scgRNA-F_D is formed by the annealing of two ssDNA with fluorescent and inhibitory groups and one sgRNA. When the bulge part is cleaved by the trans-activated Cas12a, the DNA dissociates from the RNA and the DNA strand breaks. The distance between the fluorescence group and the quenching group becomes larger, and the quenching group loses its inhibition on the fluorescence group. We use 492nm and 518nm as the excitation wavelength and reception wavelength of the FAM group respectively. The fluorescence activity is activated and released, and the data are recorded and read out by the enzyme labeling instrument as the indication information of the nucleic acid signal. At the same time, the released sgRNA combines the unoccupied Cas12a to amplify the signal exponentially.
Reference
[1]Shi K, Xie S, Tian R, et al. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci Adv. 2021;7(5):eabc7802. Published 2021 Jan 27.
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