Part:BBa_K5124039
Cas13a PLAUR 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 PLAUR 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 24-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 wiki) looked for sequences 25-nucleotides long where the 25th nucleotide was non-G. The resulting 24-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_K5124022), 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.
The respective target part is BBa_K5124028.
Results
Results from in-vitro transcription reactions are shown below.
All columns on the RNA tape showed there was RNA present around the expected length for sgRNA, and target RNA.
Due to the small size of the sgRNA sequence and the upper marker in the sample buffer being degraded, the length of RNA sequences on this graph will not be accurate. (See Figure above showing uniformity among variants of each RNA component.) A 52 nucleotide sgRNA peak was expected, the peak at 41 nucleotides demonstrates that a short RNA was transcribed. We were happy that this demonstrates successful transcription of our RNA. A peak at 119 nucleotides may indicate that some template DNA was not cleaved (see protocol on Wiki) and transcription carried on past the end of the sgRNA sequence. There is also remains of a degraded upper marker from the BR sample buffer. The concentration of PLAUR sgRNA was determined to be 18.9 ng/ul which was a usable concentration for our final tests.
We mixed our Cas13a, each sgRNA, the corresponding target RNA and our fluorescent probes and saw an increase in fluorescence with time for PLAUR, CXCL8, RGS16 and NR4A1. This 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.
We ran a series of control experiments. Four negative controls left out one component from the reaction. One control included the wrong target demonstrating that the specific sgRNA/target combination was required. The miss folded FOSB sgRNA sequence was beneficial as it demonstrates that the sgRNA must fold correctly to activate Cas13a.
Conclusion
We successfully expressed our Cas13a enzyme and achieved reasonable purity. We can tell the Cas13a system worked because we got positive results when the Cas13a, sgRNA, target and probe were added together (as shown in the graph above), but also because we had negative controls that caused no reaction. These were:
The system missing one of the 4 parts showing all parts are necessary.
The system with the wrong target showing that the system only activates with the correct RNA sequence.
The system with the spacer FOSB which folded incorrectly, showing the testing for the folding of sequences was necessary.
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
Sequence and Features
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