Fig. 5.

Base editing of genomic DNA in human cells by Sa-sgCBE. a Cartoon representation of the comparison between SpCas9 sgRNA and SaCas9 sgRNAs. The upper cartoons were the crystal structure of the SaCas9 sgRNA scaffold. Below cartoons represented the comparison between the SaCas9 sgRNA scaffold and the SpCas9 sgRNA scaffold. Loops of each sgRNAs were shown in red. b SaCas9 sgRNA sequence and schematic diagram of MS2-modified sgRNA scaffold. (Blue box enclosed the 3rd stem-loop that was deleted when resolving the crystal structure). c Efficiency of cytosine editing with Sa-sgCBE. HEK293T cells were transfected with plasmids expressing BE3-SaKKH or Sa-sgCBEs targeting a set of three different sites. C-to-T editing efficiencies were analyzed by Sanger sequencing and EditR calculating. Each experiment was repeated at least three times. d Schematic diagram showing AAV vectors encoding Sa-sgCBE, one expressing SaCas9 nickase and the other one expressing U6-sgRNA and MCP-APOBEC1-UGI. e Efficiency of cytosine editing with AAV-encoded Sa-sgCBE. HEK293T cells were transduced with Sa-sgCBE AAVs targeting HEK4 site at the indicated multiplicity of infection (MOI), with or without bortezomib treatment. Four days later, cells were harvested and subjected to DNA analysis