Difference between revisions of "Part:BBa K5124035"

Revision as of 22:24, 29 September 2024

Cas13a CXCL8 sgRNA

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.

In 2021 McLoughlin et al. published RNA-Seq data from cattle infected with bTB at several timepoints during the disease progression [1]. They identified 19 potential biomarkers that were present across the entire length of the infection time course. We would have liked to have tested all 19 sequences but after two rounds of the design-build-test-learn cycle (see our Wiki) we focused on:
CXCL8- chemokine ligand 8, involved in infection response and tissue injury.
FOSB- FBJ murine osteosarcoma viral oncogene homologue B, plays a role in regulating cell proliferation, differentiation and transformation.
NR4A1- nuclear receptor subfamily 4, group A, member 1, plays a role in inflammation and apoptosis.
PLAUR- plasminogen activator, urokinase receptor, a biomarker of inflammation.
RGS16- regulator of G-protein signalling 16, linked to many different disease states.

This composite 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 the CXCL8 spacer sequence that is 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-nucleotide spacer sequence free to bind to the target RNA (Figure 1 a). In addition, the T7 promoter adds three G nucleotides to the 5’ end of the transcript. In this case addition of these G’s leads to the IPKnot++ software predicting an additional loop in the spacer sequence (Figure 1 b) however you will see in the results section this has not prevented this spacer from binding to its target RNA.

Design and Characterisation

This crRNA sequence was taken from the paper by Kelner et al. [4].

We used the Bos taurus genome sequence from a Hereford cow (BioProject accession number PRJNA450837) as a starting point to design our spacer sequences.

The DNA sequences of the 19 targets were downloaded and using the associated annotations the introns were removed using the splicing function in SnapGene. The resulting mRNA sequences were inputted into a Python script that screened for potential spacer sequences. The Cas13a-CRISPR system requires that the protospacer flanking sequence (PFS), the adjacent nucleotide to the 3’ end of the target site, must be a non-guanine. Therefore, the Python script (see GitHub) looked for sequences 21-nucleotides long where the 21st nucleotide was non-G. The resulting 20-nucleotide spacer sequences that included either a CCCC or GGGG repeats were filtered out, as the presence of these would cause misfolding with the crRNA hairpin loop. In addition, sequences with more than one uracil base were filtered out, as uracil bases easily bind to other RNA nucleotides. The Cas13a crRNA sequence was appended to the 5’ end of each spacer sequence which were then analysed for secondary structure using the on-line software IPknot++ [5]. Final spacer sequences were chosen by the highest minimum free energy, denoting least intra-sequence binding within the spacer region, and minimum inter-sequence binding between the spacer and crRNA sequences.

Due to the minimum synthesis length of 125 base pairs for IDT gBlocks, this composite part was synthesised containing: a 5’ crRNA sequence (BBa_K5124012), a 3’ spacer sequence (BBa_K5124018), 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 [6]) carrying an ampicillin selection marker.

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. Sato K, Kato Y. Prediction of RNA secondary structure including pseudoknots for long sequences. Brief Bioinform. 2022 Jan 17; 23(1).

6. 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