RNA

Part:BBa_K4664005

Designed by: Yiming Dong   Group: iGEM23_HS-China   (2023-10-09)
Revision as of 07:15, 12 October 2023 by Chrisdong (Talk | contribs)


crRNA

The crRNA guides the Cas protein to the target DNA sequence for precise genome editing or other DNA manipulation purposes.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[05]

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.
p464005-1.png
Figure 1. 1 pM RNP.
p464005-2.png
Figure 2. 10 pM RNP.
p464005-3.png
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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