Difference between revisions of "Part:BBa K5124001"

Revision as of 13:00, 21 August 2024

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.

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 [1, 2]. 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 [3]. 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 [4]. 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.

Characterisation

The coding sequence for LwCas13a was taken from the plasmid pC029-Lw2Cas13a from Leptotrichia wadei F0279 (Addgene plasmid #91919) [5] 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 [6]) carrying an ampicillin selection marker with: the T7 promoter BBa_I719005, the RBS from bacteriophage T7 gene 10 (similar to BBa_K1758100) and the transcription terminator from bacteriophage T7 RNA polymerase BBa_K731721.

The LwCas12a expression plasmid was transformed into E. coli BL21(DE3) (Novagen) and protein expression was induced by autoinduction media [7]. The enzyme was purified via Ni-affinity and gel filtration chromatography. Please see our Wiki for the detailed protocol (Wiki experiments).

Results

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References

[1] 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.

[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] 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.

[4] 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.

[5] 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.

[6] Sutcliffe JG. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 1:77-90.

[7] Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005 May; 41(1):207-34.

===Sequence and Features