Part:BBa_K5124012
Cas13a crRNA
Usage and Biology
The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.
This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type VI CRISPR loci of Leptotrichia wadei [3]. This sequence is combined with one of five spacer sequences that are complimentary to our target bovine RNA. Once transcribed into RNA, the 29-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LwCas13a, leaving the 20/24-nucleotide spacer sequence free to bind to the target RNA (Figure 1). In addition, the T7 promoter adds three G nucleotides to the 5’ end of the transcript.
Design and Characterisation
This crRNA sequence was taken from the paper by Kelner et al. [4]. Due to the minimum synthesis length of 125 base pairs for IDT gBlocks, this basic part was synthesised as part of one of five composite parts (BBa_K5124035 to BBa_K5124040) each containing: a 3’ spacer sequence (BBa_K5124018 to BBa_K5124023), 5’ T7 promoter (BBa_K5124041) and Type IIS compatible prefix and suffixes. The gBlock was cloned into a high copy plasmid (origin of replication from pUC18 [5]) carrying an ampicillin selection marker.
Results
We mixed our Cas13a, an sgRNA, the corresponding target RNA and our fluorescent probes and saw an increase in fluorescence with time for PLAUR, CXCL8, RGS16 and NR4A1. Tis shows that when activated by the correct sgRNA Cas13a binds to the target and cleaves the probe. However, there was no increase in fluorescence for FOSB. This is because we discovered a mistake in the folding of the FOSB sgRNA. However this proved beneficial as it shows that if the sgRNA does not fold correctly Cas13a cannot cleave the probes.
Please see composite parts:
K5124035
K5124036
K5124037
K5124039
K5124040
for further usage and results.
Conclusion
Because of these results, we can determine that our Cas13a system would function with these specific targets and spacers, but also future teams could use the system themselves by finding their own spacer sequences (and respective targets) and testing the folding. These spacer parts could then be combined with this crRNA to develop sgRNA, which could be combined with our Cas13a protein to make their own functioning system.
References
1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.
2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.
3. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017 Oct 12; 550(7675):280-4.
4. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019 Oct; 14(10):2986-3012.
5. Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.
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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