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.
This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type VI CRISPR loci of Leptotrichia wadei [1]. 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 double hairpin loop, which is recognised and bound by LwCas13a, leaving the 20-nucleotide spacer sequence free to bind to the target RNA (Figure 1).
Characterisation
This crRNA sequence was taken from the paper by Kelner et al. [2]. It was synthesised by IDT 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_I719005 and BioBrick compatible prefix and suffixes. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.
Please see composite parts:
K5124035
K5124036
K5124037
K5124038
K5124039
K5124040
for further usage and results.
References
[1] 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.
[2] 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.
[3] 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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