Fig. 3: Restoring the nuclease activity SpdNG-LWQT toward 5′-TGG-3′ and 5′-CAT-3′ PAMs.

a In vitro DNA cleavage assay for Cas9 activity. Purified his₆-tagged Cas9 proteins were incubated with in vitro transcribed sgRNA and linearized DNA targets, and the reaction was performed at 37 °C. b DNA cleavage by SpCas9wt and SpNG-LWQT with sgegfp targeting 5′-TGG-3′ or 5′-CAT-3′ PAM. DNA target with SpCas9wt but without any sgRNA was served as the negative control. Representative image from two independent repeats. c In vivo plasmid DNA cleavage assay with SpdCas9wt (blue) and SpNG-LWQT (orange). Plasmid pCS-Plpp1-SpCas9wt or pCS-Plpp1-SpNG-LWQT was transferred into E. coli BW25113(F′) harboring pZE-eGFP-sgegfp containing sgegfp targeting 5′-TGG-3′ or 5′-CAT-3′ PAM targets on eGFP. Data indicated the mean ± standard deviation (n = 3 independent biological replicates). d In vivo chromosomal DNA cleavage assay. Plasmid pCS-Plpp1-SpCas9wt or pCS-Plpp1-SpNG-LWQT was transferred into E. coli BW25113(F′)::eGFP harboring pZE-sgegfp containing sgegfp targeting 5′-TGG-3′ or 5′-CAT-3′ PAM targets on chromosomally integrated eGFP. Cells were plated on Luria-Bertani (LB) plates containing 0.5 mM IPTG and appropriate antibiotics. Negative controls (−) were transformed with empty pCS27 plasmid. Representative image from two independent repeats. Source data are provided as a Source Data file.