Fig. 5.

Examination of the StCas9 system in human 293T cells. a Comparison of different sgRNA expressing strategies in 293T cells. Transfection assay was conducted with the hStCas9 expression vector and the surrogate reporter pRep.eGFP.SP1.PAM5 combining with different vectors for the wild type sgRNA design. For the linearized pRep.eGFP control, the reporter plasmid was linearized with BamH I before transfection. b Functional analysis of the SP1.sgRNA.Opti design for guiding StCas9 activity in 293T cells. Transfection assay was conducted with corresponding SP1.sgRNA.Opti and hStCas9 expression vectors and the surrogate reporter pRep.eGFP.SP1.P5M4. c Validation of the StCas9 activity on targets from mouse ROSA26 locus in 293T cells. Transfection assay was carried out with the sgRNA-StCas9 expression vectors (mU6.sgRNA.Opti-CMV.hStCas9) and corresponding mammalian cell surrogate reporters, pRep.eGFP.R26A/B/C