Difference between revisions of "Team:Cardiff Wales/Description"
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| − | <p>Dr. Daniel Pass | + | <p>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. |
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| + | Here, we targeted the <i>E. coli</i> 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 <a href="http://github.com/passdan/scriptdrop/blob/master/cas9_targeter.py">Python script</a> developed to find appropriate paired regions from a FASTA formatted genomic DNA region for paired target sequences using <a href="http://www.clontech.com/GB/Products/Genome_Editing/CRISPR_Cas9/Resources/Designing_sgRNA">guidelines</a> from Takara Bio USA alongside additional sources. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
</div> | </div> | ||
Revision as of 16:13, 19 October 2016
'Cas-Find' is a novel bioluminescent system for point-of-care diagnostic testing.
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
Proof of concept in vitro system targeted to the Escherichia coli 16S rRNA locus.
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
Title
Islo lorem etc
Fig 3. Summary of Characterisation
