Part:BBa_K5124001
LwCas13a codon opt
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 Cas13a enzyme is from Leptotrichia wadei and was chosen for our project as it was used in the SHERLOCK detection system developed by Gootenberg et al. 2017 and later Kellner et al. 2019 [3, 4]. Although RNA guided, in contrast to the widely known Streptococcus pyogenes Cas9, LwCas13a recognises and specifically cleaves RNA, therefore we are developing an RNA detection system with this enzyme.
Originally named C2c2 (class 2 candidate 2), Cas13a was first identified in Leptotrichia shahii [5]. It is part of a class II, type VI CRISPR system that does not use a trans-activating (tracrRNA) but instead the Cas13a enzyme itself can cleave pre-crRNA [6]. Each mature crRNA consists of a consensus repeat sequence and a 20-nucleotide spacer sequence. The repeat sequence BBa_K5124012 folds into a hairpin loop that is recognised and bound by Cas13a. The spacer sequence is complimentary to a sequence of viral RNA that the bacterium has previously been exposed to.
Design and Characterisation
The coding sequence for LwCas13a was taken from the plasmid pC029-Lw2Cas13a from Leptotrichia wadei F0279 (Addgene plasmid #91919) [7] and codon optimised for expression in E. coli using the IDT tool. Any restriction enzyme sites that would prevent compatibility with BioBrick and Type IIs cloning were removed. A 6xHis tag and TEV protease cleavage site was added at the N-terminal, Type IIS cloning prefix and suffixes were added and the complete sequence was synthesised as a g-Block by IDT. This was cloned into a medium copy plasmid (origin of replication from pBR322 [8]) carrying an ampicillin selection marker with a composite part comprising the T7 promoter, lac operator and the RBS from bacteriophage T7 gene 10 (BBa_K5124042) and the transcription terminator from bacteriophage T7 RNA polymerase (BBa_K395601)
The LwCas13a expression plasmid was transformed into E. coli BL21(DE3) (Novagen) and protein expression was induced by autoinduction media [9]. The enzyme was purified via Ni-affinity and size exclusion chromatography. Please see our Wiki for the detailed protocol (Wiki experiments).
Results
We used this Cas13a sample in our final test of the overall system. Purity could still be improved for a real life test. Cas13a bands on western blots look clearer and show lower levels of impurities than our first purification. Using Qubit fluorometer 2.0, we measured the concentration of the protein samples and the first purified Cas13a sample was 612 µg/ml whilst last purified Cas13a sample was 183 µg/ml. The significant decrease in the concentration of the samples, along with the SDS-PAGE results demonstrates that although protein concentration in the sample decreased, purity increased.
Cas13a proteins were purified from crude cell lysate using His-Trap (Cytiva) columns, and AKTA Pure (Superdex 200 10/300 120mL Cytiva) Size Exclusion Chromatography (SEC). The resulting SDS-Page gel was photographed and analysed to determine the success of purification following each technique. To quantify purity of Cas13a, the following analysis was used:
The image was cropped to the relevant lanes, (shown in yellow on Figure 2a) with the band containing the Cas13a protein also separately cropped (shown in red on Figure 2a).
These images were saved as .png files named img1-4.
A custom Matlab script (on our wiki) was used to import each image, convert to a numerical matrix of RGB intensities, and count blue pixels given a certain threshold (Red <=100, Green <=100, Blue >= 100).
Final values were plotted (Figure 2b)
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.
For more detail on these results, see our Engineering Success
Conclusion
We successfully expressed our Cas13a enzyme and achieved reasonable purity, as although the concentration of protein decreased in the final purification there were fewer bands on the SDS-PAGE gel. 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 Figure 3 above), but also because we had negative controls that caused no reaction (Figure 4). 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.
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 sgRNA parts could then 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. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 28; 356(6336):438-42.
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. Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, et al. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell. 2015 Nov 5; 60(3):385-97.
6. East-Seletsky A, O'Connell MR, Knight SC, Burstein D, Cate JH, Tjian R, et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature. 2016 Oct 13; 538(7624):270-3.
7. 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.
8. Sutcliffe JG. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 1:77-90.
9. Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005 May; 41(1):207-34.
Sequence and Features
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- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2114
Illegal BglII site found at 3266 - 23COMPATIBLE WITH RFC[23]
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