Part:BBa_K5124032
Cas12a RD4_a 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 2007 Taylor et al. demonstrated that it was possible to detect bTB DNA in cattle lymph nodes [2]. One the targets for their assay was RD4 which is known as a region of difference between different subspecies of tuberculosis causing bacteria. Therefore, the RD4 region has the potential to be used to specifically detect bTB. Importantly, RD4 is not present in the attenuated BCG strain, which is used for vaccination, therefore this detection of this target would be of potential use for a diagnostic capable of differentiating infected and vaccinated animals (DIVA).
This composite part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type V CRISPR loci of Lachnospiraceae bacterium ND2006 [3]. This sequence is combined with the RD4_a spacer sequence that is complimentary to our target bovine TB DNA. Once transcribed into RNA, the 21-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA (Figure 1). In addition, the T7 promoter adds three G nucleotides to the 5’ end of the transcript.
Design and Characterisation
The crRNA sequence was taken from the paper by Moreno-Mateos et al. [4].
We used the Mycobacterium bovis AF2122/97 genome sequence (Accession number LT70834) [5] as a starting point to design our spacer sequences.
The DNA sequence coding for RD4 was downloaded and inputted into a Python script that screened for potential spacer sequences. The Cas12a-CRISPR system requires that the protospacer-adjacent motif (PAM), the four nucleotides 3’ to the end of the target site, must be TTTV. Therefore, the Python script (see GitHub) looked for sequences 24-nucleotides long where final four nucleotides were either TTTA, TTTC or TTTG. The resulting 20-nulceotide 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 Cas12a 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++ [6]. 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_K5124011), a 3’ spacer sequence (BBa_K5124015), 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 [7]) 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. 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.
4. 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.
5. Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoor H, Pryor M, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A. 2003 Jun 24; 100(13):7877-82.
6. Sato K, Kato Y. Prediction of RNA secondary structure including pseudoknots for long sequences. Brief Bioinform. 2022 Jan 17; 23(1).
7. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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