Fig. 3: dCas9- and Cas9-based SNP-Chip detection of the SCD-associated SNP from genomic DNA samples.

a, Schematic of SNP-Chip functionalized with the dCas9–gRNA complex. gRNA-HTYa was used. In the presence of the SCD-associated SNP, dCas9-HTYa does not hybridize completely with its DNA target, and the DNA dissociates from the dCas9–gRNA complex. b, Schematic of SNP-Chip functionalized with the nuclease-active Cas9–gRNA complex. gRNA-HTYa was used. c, Real-time C response of dCas9-HTYa in the presence of HbAA and HbSS patient genomic samples. The black arrows indicate sensor rinsing. d, Endpoint C response of the dCas9-HTYa construct in the presence of HbAA and HbSS patient genomic DNA samples (60 ng µl⁻¹) (t = 5.849; d.f. = 15; n ≥ 8 technical replicates). e, Endpoint C response of SNP-Chip with the nuclease-active Cas9-HTYa construct after incubation with homozygous HbAA and HbSS patient genomic DNA samples (60 ng µl⁻¹) (t = 2.867; d.f. = 15; n = 8 technical replicates). f, Schematic of the sensitivity test during which the nuclease-active Cas9-HTYa construct was incubated with varying concentrations of target HbAA DNA samples (10–60 ng µl⁻¹). g, Results of the Cas9-HTYa construct sensitivity test, during which the nuclease-active Cas9-HTYa construct was incubated with varying concentrations of target HbAA DNA samples (10–60 ng µl⁻¹). Each point in the scatter plot shows a mean value ± s.d. (n ≥ 5 technical replicates). h, Results of blind studies of the C response of SNP-Chip functionalized with nuclease-active Cas9-HTYa in the presence of patient samples with each phenotype (healthy or SCD) (t = 3.325; d.f. = 10; n = 3 biological replicates, with two technical replicates per biological sample). In d, e and h, significance was determined by unpaired t-test (*P < 0.05; **P < 0.01; ****P < 0.0001; all two tailed). All box and whisker plots show the minima, Q2, median, Q3 and maxima.