Part:BBa_K5124000
LbCas12a 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 Cas12a enzyme is from Lachnospiraceae bacterium ND2006 and was chosen for our project as it was used in the DETECTR detection system developed by Chen et al. 2016 [3]. Similar to the widely known Streptococcus pyogenes Cas9, LbCas12a is an RNA guided enzyme that recognises and specifically cleaves DNA, therefore we are developing a DNA detection system with this enzyme.
Originally named Cpf1 (CRISPR-Cas loci of Francisella novicida U112), Cas12a was identified in L. bacterium ND2006 by Zetsche et al. [4]. It is part of a class II, type V CRISPR system that does not use a trans-activating (tracrRNA) but instead the Cas12a enzyme itself can cleave pre-crRNA. Each mature crRNA consists of a consensus repeat sequence and a 20-nucleotide spacer sequence. The repeat sequence BBaK5124011 folds into a hairpin loop that is recognised and bound by Cas12a. The spacer sequence is complimentary to a sequence of viral DNA that the bacterium has previously been exposed to.
Characterisation
The E. coli optimised coding sequence for LbCas12a was taken from the plasmid LbCpf1-2NLS (Addgene plasmid #102566) [5]. 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 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 LbCas12a 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 size exclusion chromatography. Please see our Wiki for the detailed protocol (Wiki experiments).
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. Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018 Apr 27; 360(6387):436-9.
4. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22; 163(3):759-71.
5. Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, et al. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun. 2024 Dec 8; 8:1-9.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1461
Illegal BglII site found at 2198
Illegal BglII site found at 3210 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3112
- 1000COMPATIBLE WITH RFC[1000]
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