Part:BBa_K2961003
gRNA / crRNA fixed-loop repeat for Cpf1 / Cas12a
Sequence (crRNA repeat) that all Lba Cas12a gRNAs need at the beginning. Also known as fixed loop
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
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).
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), which were proofed right by our model.
【fig.2】Cas12a test strip result
【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].
【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.
Characterization by iGEM22_Worldshaper-HZ
- Group: iGEM22_Worldshaper-HZ
- Author: Haoyu Xu
- Summary:Characterization of BBa_K2961003 working efficiently as a Cas12a handle region in three guide RNAs for a CRISPR-Cas12a system.
We obtained this part from iGEM19_CMUQ team. In this experiment, our team has tested and verified the efficiency of part BBa_K2961003 designed by iGEM19_CMUQ.[ https://parts.igem.org/Part:BBa_K2961003] We incorporated this sequence into three guide RNAs, including igRNA (AAG+GAPDH as guide+ hsa_circ_0001982 as trigger) (BBa_K4409009), gRNA (AAG+ BBa_K3859000) (BBa_K4409015), and gRNA (AAG+ GAPDH as guide)(BBa_K4409016), and used it as the binding site with Cas12 enzyme. By using a CRSPR-Cas12 system for these guide RNAs, our experiments showed that BBa_K2961003 was sensitive as a Cas12a handle region for these guide RNAs.
Experiment results
BBa_K2961003 in igRNA (AAG+GAPDH as guide+ hsa_circ_0001982 as trigger)
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]
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