DNA

Part:BBa_K5124012

Designed by: Louise Brown   Group: iGEM24_Exeter   (2024-08-19)
Revision as of 21:35, 24 September 2024 by Lb1028 (Talk | contribs)


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 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).

Figure 1: Cas13a crRNA folded into the double hairpin loop

Characterisation

This crRNA sequence was taken from the paper by Kelner et al. [4]. 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 [5]) carrying an ampicillin selection marker.

Please see composite parts:
K5124035
K5124036
K5124037
K5124038
K5124039
K5124040
for further usage and results.

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


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]


[edit]
Categories
Parameters
None