Difference between revisions of "Team:Cardiff Wales/Description"

Laboratory-based tests for sexually transmitted infections (STI), such as nucleic acid amplification (NAA) , require specalised infrastructure, equipment and procedures for optimal performance ^[2]. As such the utilisation of laboratory-based tests is generally expensive and time consuming, limiting the availability of results for immediate use in management decisions, potentially impacting on patient prognosis. In addition the majority of STI testing is conducted in resource-constrained environments, where such infrastructure and facilities are unavailable ^[3].

Most bacteria and archaea possess RNA-mediated adaptive defence mechanisms composed of clustered regularly interspaced short palindromic repeats (CRIPSR) and CRISPR-associated proteins (Cas). Exogenous sequences are incorporated into the bacterial or archaeal genome at the CRISPR loci, transcription through which produces CRISPR RNA (crRNA). An additional trans-activating RNA (tracrRNA) is required for interference, and is partially complementary to the crRNA. In Streptococcus pyogenes this forms a crRNA:tracrRNA ^[5] duplex which recruits the endonuclease Cas9, and collectively these molecules achieve sequence specific DNA interrogation and cleavage. This is summarised in Figure 1, demonstrating the sequential acquisition of exogenous DNA, RNA processing and interference. In synthetic applications a single guide RNA (sgRNA) construct fulfills the role of the crRNA:tracrRNA duplex. This sgRNA consists of a 20 nucleotide sequence complementary to the target, a 42 nucleotide Cas9 binding RNA structure, and a 40 nucleotide transcription terminator ^[6]. We have developed a novel detection system for point-of-care testing (POCT) utilising the capability of CRISPR/Cas9 to achieve sequence specific interrogation.

Luciferases catalyse bioluminescent reactions using ATP in the presence of molecular oxygen (O₂) and luciferin (LH₂), with in vivo and in vitro applications in imaging and detection. Wild-type luciferases are highly sensitive to pH and are thermolabile, undergoing inactivation and bathochromic shift at 25^oC ^[7]. We fused the C- and N- terminal fragments of a thermostable pH-tolerant Photinus pyralis luciferase mutant to a dCas9 isoform optimized for expression in E. coli. dCas9which lacks catalytic activity, and as such targeted sequences do not undergo cleavage. sgRNA constructs target these chimeric proteins to adjacent sequences, resulting in the reconstitution of luciferase activity and bioluminescence in the prescence of luciferin. The reconstitution of bioluminesce significantly greater than background activity constitutes the positive signal for pathogen detection. The mechanism of this 'Cas-Find' system is demonstrated in Fig 2.

An sgRNA construct for our proof-of-concept were expressed in pSBC13 under a T7 promoter and the control of the lac operator. An additional sgRNA construct was expressed in the pCOLADuet. The design of these constructs is discussed in more detail below.

Dr. Daniel Pass developed a program to assist in designing sgRNA constructs for non-standard genomic regions and species, to the specifications required for this system. Here, we targeted the  E. coli 16S rRNA locus in order to facilitate proof-of-concept testing of our in vitro system. The design of these constructs was achieved using a Python script developed to find appropriate paired regions from a FASTA formatted genomic DNA region for paired target sequences using guidelines from Takara Bio USA alongside additional sources.The script initially identifies proto-spacer adjacent motif (PAM) sequences (5'-NGG-3') in this FASTA sequence in forward and reverse. The sgRNA sequence is complementary to the 20 nucleotides upstream of the PAM sequence, after accounting for other enhancement features. Viable pairs within a defined range of each other are selected and passed to BLASTn to test for simple alignment against a reference dataset. This would include the remainder of the species genome, and also multiple cross-reactive species. The output is a FASTA file of potential probe pairs, a table.txt file of the same information in a graphical representation, and the results of the blast search, to aid in choosing probes which do not demonstrate cross-reactivity

Fig 2. Summary of sgRNA design

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Fig 3. Summary of Characterisation