DNA

Part:BBa_K2644101

Designed by: Xinyi Wang   Group: iGEM18_TJU_China   (2018-07-26)


FnCas12a

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1169
    Illegal BglII site found at 1502
    Illegal BglII site found at 1622
    Illegal BglII site found at 1916
    Illegal BglII site found at 2360
    Illegal BglII site found at 3251
    Illegal BglII site found at 3341
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2396
    Illegal NgoMIV site found at 3235
    Illegal NgoMIV site found at 3446
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

FnCas12a is an important part in our group. It is used to add with crRNA to perform detection function. Because of both cis and trans cleavage function that cleave DNA 24 base pairs downstream from the PAM site., FnCas12a can be used to reach gene detection.

Reference

Li, Shi Yuan, et al. "CRISPR-Cas12a has both cis - and trans -cleavage activities on single-stranded DNA." Cell Research (2018).


Results

T--TJU_China--2-1.png


Figure 1. Result of protein expression and purification of FnCas12a. (A) SDS-PAGE gel of result of affinity chromatography (Ni-NTA) result. Lane M, marker. Lane 1, before washing by Buffer A. Lane 2, after washing by Buffer A. Lane 3, before elution by Buffer B. Lane 4, after elution by Buffer B. Buffer A (50 mM Tris-HCl (pH8.0), 1.5 M NaCl, 5% glycerol, 30 mM imidazole). Buffer B (50 mM Tris-HCl (pH8.0), 1.5 M NaCl,1 mM DTT and 5% glycerol, 600 mM imidazole). (B) SDS-PAGE gel of result of ion exchange. Lane M, marker. Lane 1, purified FnCas12a. (C) SDS-PAGE gel of result of gel filtration. Lane M, marker. Lane 1, purified FnCas12a. (D) The result of ion-exchange chromatography program. (C) The result of gel filtration program.


Effect of ions on FnCas12a’s collateral cleavage activity

iGEM Thrace 2021

FnCas12a is a Cas12a ortholog. It’s activity is modified in the presence of ions. Specifically, Mn2+ ions are able to trigger FnCas12a-mediated dsDNA cleavage activity. Moreover, in the presence of Mn2+ ions the CrRNA-independent DNase activity of FnCas12a is activated for both dsDNA and ssDNA.

Reference

Li, B., Yan, J., Zhang, Y., Li, W., Zeng, C., Zhao, W., Hou, X., Zhang, C., & Dong, Y. (2020). CRISPR-CAS12A possesses unconventional DNase activity that can be inactivated by synthetic oligonucleotides. Molecular Therapy - Nucleic Acids, 19, 1043–1052. https://doi.org/10.1016/j.omtn.2019.12.038

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