Selected article for: "cis cleavage and fluorescence signal"
Author: Long T. Nguyen; Brianna M Smith; Piyush K. Jain
Title: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection
  • Document date: 2020_4_14
    • ID: n5kpvsvg_6
    Snippet: While 7-mer ssDNA extensions on the 3'-end of crRNA increases trans-cleavage activity with LbCas12a, we questioned if this is consistent across other orthologs of Cas12a. To investigate further, we carried out an in vitro cis-cleavage and trans-cleavage assay of AsCas12a and FnCas12a with an extended crGFP compared to a wild-type crGFP ( Fig. 2j and Supplementary Fig. 10 ). Interestingly, the crGFP+3'DNA7 showed similar results with FnCas12a; ho.....
    Document: While 7-mer ssDNA extensions on the 3'-end of crRNA increases trans-cleavage activity with LbCas12a, we questioned if this is consistent across other orthologs of Cas12a. To investigate further, we carried out an in vitro cis-cleavage and trans-cleavage assay of AsCas12a and FnCas12a with an extended crGFP compared to a wild-type crGFP ( Fig. 2j and Supplementary Fig. 10 ). Interestingly, the crGFP+3'DNA7 showed similar results with FnCas12a; however, it exhibited an opposite effect with AsCas12a. However, the cis-cleavage activity was found to be comparable between the crGFP and crGFP+3'DNA7 for all the orthologs tested. Overall, LbCas12a showed the highest fluorescence signal, which is consistent with previous studies. 13, 18 Through observation of the fluorophore-quencher-based reporter assay and time-dependent gel electrophoresis, we hypothesized that the various extensions of ssDNA on the crRNA induce conformational changes on LbCas12a that result in enhanced endonuclease activity.

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