RNA

Part:BBa_K2927006

Designed by: KUO, PEI-CHING   Group: iGEM19_CCU_Taiwan   (2019-09-04)


guide RNA of LbCas12a

The CRISPR/Case system uses guide RNA (gRNA) and crRNAs to recognize their targets. The gRNA sequence binds to Cas12a protein, which promotes crRNA to recognize its target double-stranded DNA through sequence complementation.

Characterization by iGEM21_GreatBay_SZ

  • Group: iGEM21_GreatBay_SZ
  • Author: TianYi Huang

We attached the repeat sequence to guide RNA used to recognize their targets(Part:BBa_K3859013). The crRNA will bind with cas12a enzyme, then the target DNA will be recobgnised and unwound by cas12a. After target DNA binds with guide RNA, cas12a will be activated and cleveage the sequence around it. In our project, we primarily used Cas12a detection technology for the identification of spores. This decision was made with reference to the methodology used in the HOLMES system

Experienment and result from iGEM21_GreatBay_SZ

We used both the test strip and the florencence method in vitro. For the test strip, we successfully got the correct result for all the barcodes(fig.2). For the florencence method, we got the graphs of the florencence against time(fig.3)[2], which were proofed right by our model.

In addition, in order to test the specificity of our barcode design. We constructed 7 barcodes and their matching CRISPR RNAs (crRNAs) and assayed all permutations in vitro using cas12a florencence detection(fig.4).

T--GreatBay SZ--cas12a detection.png

【fig.1】Cas12a detection

T--GreatBay SZ--cas12a test strip.png

【fig.2】Cas12a test strip result

T--GreatBay SZ--fluorescence detection.png

【fig.3】Cas12a fluorescence detection result. On the left of the symbol "-" refers to barcode name; On the right of the symbol "-" refers to crRNA name. All results for barcode mismatch with crRNA are shown in grey. E.g. "T-T" means the mixture of T-barcode and T-crRNA.

In addition, in order to test the specificity of our barcode design. We constructed 7 barcodes and their matching CRISPR RNAs (crRNAs) and assayed all permutations in vitro using cas12a florencence detection(fig.3)[2].


T--GreatBay SZ--cas12a orthogonal experiment heat map.png

【fig.4】Heatmap of endpoint fluorescence values from in cas12a fluorescence detection of all combinations of 7 barcodes and 7 crRNAs assessing specificity of each barcode-crRNA pair.


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]


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