PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics

Figures

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  Fig. 1. Overview of the PathoGD pipeline. The main steps involved in the pangenome and k-mer modules are highlighted. Further details about each step are illustrated in Supplementary Fig. 1.

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  Fig. 2. PathoGD in silico results of pangenome and k-mer modules for five bacterial species. a Number of target-specific guide RNAs identified from the pangenome and k-mer modules in PathoGD. The k-mer (no primers) module refers to the workflow without primer design (see Supplementary Fig. 1). Results for the pangenome and k-mer modules were filtered to only observations where gRNAs and primers were present across 90% of strains in each target species, primers had at least nine mismatches to the non-target species, and at least one mismatch of gRNA to non-target genomes occur in the PAM or seed region. b Average copy numbers of gRNAs identified from the k-mer (no primer) module for the five bacterial species. Black dotted lines refer to the cut-off at 2 copies. c Unique and overlapping gRNAs identified from the pangenome and k-mer modules in (a) for five bacterial species. d Distribution of gRNAs designed with the k-mer module across the coding, noncoding, or overlapping regions within the genome. All results were generated using the ‘user_all_subsample’ workflow, with 100 genomes randomly subsampled for primer and guide RNA design for each target species, except for T. pallidum (n =…

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  Fig. 3. Experimental validation of primers and gRNAs for Streptococcus pyogenes in an RPA/CRISPR–Cas12a assay. a Distribution of guide RNAs across the S. pyogenes ATCC 19615 genome (RefSeq assembly accession GCF_000743015.1). b Fluorescence profiles of guide RNAs across 35 min for the S. pyogenes ATCC 19615 strain. Data represent the average fluorescence values of three experimental replicates. Error bars represent the standard error of the mean (SEM). The specificity of each gRNA was tested against a panel of closely related organisms and other organisms likely to be present at the site of infection. c Limit of detection of the guide RNAs using 4000, 400, and 40 copies/μL of S. pyogenes gDNA, with NTC as the negative control. Data represent the normalized fluorescence at the assay endpoint for four replicates. Samples were compared to the NTC using a one-sided t-test.

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  Fig. 4. Experimental validation of primers and gRNAs for Neisseria gonorrhoeae in an RPA/CRISPR–Cas12a assay. a Distribution of guide RNAs across the N. gonorrhoeae FA1090 genome (RefSeq assembly accession GCF_000006845.1). b Fluorescence profiles of guide RNAs across 38 min for N. gonorrhoeae FA1090 strain. Data represent the average fluorescence values of three experimental replicates. Error bars represent the SEM. The specificity of each gRNA was tested against a panel of closely related organisms and other organisms likely to be present at the site of infection. c Limit of detection of the guide RNAs using 4000, 400, and 40 copies/μL of N. gonorrhoeae gDNA, with NTC as the negative control. Data represent the normalized fluorescence at the assay endpoint for at least three experimental repeats (n = 4 for g4966, n = 3 for all other gRNA sets). Samples were compared to the NTC using a one-sided t-test.

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  Fig. 5. Experimental validation of gRNAs designed using k-mer module for Neisseria gonorrhoeae. a Distribution of six selected multi-copy gRNAs across the N. gonorrhoeae FA1090 genome. Guide RNAs are present in multiple copies throughout the genome and are denoted by their names and PAM sequence separated by an underscore. b Initial assessment of six multi-copy gRNAs on N. gonorrhoeae FA1090 gDNA in an amplification-free CRISPR–Cas12a assay. The copy number for each gRNA in the FA1090 genome is listed next to the gRNA names. Data represent average fluorescence signal across 58 min. Error bars represent SEM across three experimental repeats (two technical replicates for N. gonorrhoeae in each experimental repeat). The four gRNAs selected for pooling are indicated with an asterisk. c Heatmap of average fluorescence profiles for individual and pooled multi-copy gRNAs in an amplification-free CRISPR–Cas12a assay using four selected gRNAs. NGC refers to the control with no guide RNA added. Fluorescence values were averaged across multiple experimental repeats (n = 3 for porA, n = 6 for all other gRNA sets). The presence–absence profiles of individual gRNAs across all individual and pooled…