Laboratory-based tests, such as nucleic acid amplification (NAA) or culture, require special methods of specimen transport, alongside specalised equipment and procedures for optimal performance ^[2]. As such the utilisation of laboratory-based tests is generally expensive in terms of equipment, reagents, infrastructure and maintenance. This limits 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 laboratory facilities are unavailable ^[3].

Fig 1. Summary of Cas-Find project

Dr. Daniel Pass designed sgRNA constructs targeted to 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 by Dr. Pass to test FASTA formatted genomic DNA for paired target sequences using guidelines from Takara Bio USA alongside additional sources. This script initially identifies a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3') in this FASTA sequence. The sgRNA sequence is complementary to the 20 nucleotides upstream of the PAM sequence. This is passed to BLASTn to test for simple alignment against the reference dataset, which could include the remainder of the species genome, or multiple cross-reactive species. The output is a FASTA table of potential probes, and a table.txt file of the same information in a graphical representation.

Fig 2. Summary of sgRNA design

Fig 3. Summary of Characterisation

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