Fig. 2

Multiplex nucleotide editing using SpCas9 and sgRNA variants. a High-fidelity Cas9 variants with their specific mutation sites used in this study. The crystal structure of SpCas9 protein was download from the structure resource of NCBI (PDB ID: 6O0Y). b Schematic representation of modified sgRNA. The polyU mutation was marked with brown and the introduced nucleotides in the extend loop were highlighted in red. N20 with purple indicated targeted DNA. c Comparation of C to T editing efficiency of four SpCas9 variants directed by tRNA-sgRNA and tRNA-modified sgRNA in pBE system. Two independent transformations were carried out to repeat the editing efficiency. Substitution efficiency was defined as the percentage of calli containing C to T conversion at any position in the editing targets. Samples with significant increasement were marked out using the black line. d Comparation of random mutation efficiency of four SpCas9 variants directed by tRNA-sgRNA and tRNA-modified sgRNA at three targets. Two independent transformations were carried out to repeat the editing efficiency. Indel frequency was defined as the percentage of calli containing deletion or insertion in the editing targets. Samples with significant increasement were marked out using the black line