Part:BBa_K4664005
crRNA
The crRNA guides the Cas protein to the target DNA sequence for precise genome editing or other DNA manipulation purposes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Background
crRNA is a component of the ribonucleoprotein (RNP) used in our MB-ERC2 system. It is responsible for guiding the RNP toward the amplicon and for activating Cas12a trans-cleavage via binding to complementary sequences of the Cas12a detection module on the padlocks, which cuts the link between fluorophore and quencher, releasing fluorescence signals.
Design
Sequence:
UAAUUUCUACUAAGUGUAGAUCGUCGCCGUCCAGCUCGACC
crRNA contains a hairpin (ACU-A-AGU) and a sequence identical to the Cas12a detection module on our padlocks (aside for the T-U difference in DNA-RNA).
Results
crRNA is an integral part of all our MB-ERC2 reactions. Specifically, it is connected with RNP concentrations. Figures 1-3 are results from RNP concentration trials carried out with 1 pM and 10 pM miRNA.
Figure 1. 1 pM RNP.
Figure 2. 10 pM RNP.
Figure 3. RNP finals values.
References
Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection method using SplintR Ligase. Nucleic Acids Research, 44(13), e116. https://doi.org/10.1093/nar/gkw399
He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-023-01033-1
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