Fig. 5: Experimental validation of gRNAs designed using k-mer module for Neisseria gonorrhoeae. | Communications Biology

Fig. 5: Experimental validation of gRNAs designed using k-mer module for Neisseria gonorrhoeae.

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

Fig. 5

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 combinations tested and their cumulative copy numbers in the FA1090 genome are annotated on the left of the heatmap.

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