Fig. 6: GBS Cas9 targeted mutagenesis design and phenotypic features. | Communications Biology

Fig. 6: GBS Cas9 targeted mutagenesis design and phenotypic features.

From: Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects

Fig. 6

We used allelic exchange mutagenesis approaches to generate a Δcas9 gene deletion mutant and PAM scanning deficient scas9 with R1339A and R1341A in CNCTC 10/84 and A909 (A, B). The Cas9 variants showed equivalent growth kinetics in tryptic soy broth (C, n = 3 biological replicates, each performed in technical triplicates; data points and error bars are means across biological triplicates and standard deviations at each time point). p3015b with a targeting protospacer (PS) directed against the cyl operon is incompatible with WT GBS, in which it causes lethal double stranded DNA cleavage, but it can be introduced into dcas9, scas9, and Δcas9. p3015b with a nontargeting sham protospacer can be successfully introduced into all Cas9 variants (D). Each transformation was performed in triplicate biological replicates, from which all data are shown. Confirmatory hemolysis assay results in both strains matched phenotype expectations from the regulatory model (E, n = 4 biological replicates from which all data are shown, ****p < 0.001, one-way ANOVA with Bonferroni correction).

Back to article page