MOESM1 of Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

Additional file 1:Figure S1. Base editing results of SpCas9-pBE system. Genes, target sequences and sequencing results of six editing targets were showed in SpCas9-pBE system. PAM sequence was highlighted in green, the C to T conversion bases were highlighted in blue. Red arrow indicated the mutation peak. The results were from 15 positive calli. Figure S2. Base editing results of SpCas9-rBE system.Genes, target sequences and sequencing results of four editing targets were showed in SpCas9-rBE system. PAM sequence was highlighted in green, the C to T conversion bases were highlighted in blue. Red arrow indicated the mutation peak. The results were from 15 positive calli. Figure S3. On-target activities of high fidelity SpCas9 variants guided by wild-type sgRNA.a C-T substitution frequency of SpCas9-pBE and high fidelity Cas9 pBEs at three genomic targets. b Random mutation frequency of SpCas9 and three variants at three genomic targets. All the frequencies were calculated among 15 positive calli. Figure S4. C-T substitution and random mutation occurred at NRT1.1B site in pBE system. Three samples with random mutations were selected from the calli of SpCas9-pBE and eSpCas9(1.1)-pBE complexed with the modified sgRNA each to detect the actual mutation types. PCR product were cloned to pEASY-B vetor and 27 positive clones were sent for sequencing. Arrows indicated the substitution base. Figure S5. Random mutation frequency at NRT1.1B site in pBE system. Three samples with random mutations were selected from the calli of SpCas9-pBE and eSpCas9(1.1)-pBE complexed with the modified sgRNA each. Proportions of C-T substitution (SNP), random mutation (Indel), and substitution mixed with random mutation (SNP + Indel) were shown from 27 positive B-vector cloing above. Figure S6. Schematic diagram for the constructions of base editors. Fragments, backbones and related restriction enzymes used in the construction of SpCas9-pBE (a) and SpCas9-rBE (b) base editors. Figure S7. Essential sequences used in this study.