Figure 1
From: Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9
SaCas9 induced mutation in tobacco.
(a) Expression construct for SaCas9 (top) and SpCas9 (bottom) in tobacco. A 3 × FLAG tag and 3 × NLS peptide were translationally fused in tandem to the N-terminus of Cas9. The AtADH 5′-UTR and HSP terminator enhance transcription of the Cas9 gene. NLS: nuclear localization signal. AtADH 5′-UTR: 5′ untranslated region of Arabidopsis thaliana ALCOHOL DEHYDROGENASE gene. HSP-ter: the terminator region of Arabidopsis thaliana HEAT SHOCK PROTEIN 18.2 gene. (b,c) Mutations detected in NtPDS (b) and NtFT4 (c) gene loci. The wild-type sequence is shown at the top with the target sequence underlined and the PAM in bold. DNA deletions and insertions are shown in dashes and lower case red letters. The net change in length and the number of clones are noted to the right of each sequence (+, insertion; −, deletion; ×, number of clones). (t): Genomic DNA derived from N. tomentosiformis, (s): genomic DNA derived from N. sylvestris. (d) Phenotype of pds mutant. Left A regenerated plant with wild-type PDS gene, right a regenerated plant with mutation in all four NtPDS genes. (e) Potential off-target genes for NtFT4. The target sequence of the NtFT4 gene is shown at the top with PAM. The mismatched bases in NtFT1 and NtFT2 genes are shown in blue letters. (f) CAPS analysis of NtFT4 gene locus in the T1 generation. Each T1 progeny was segregated from biallelic mutant (T0) or monoallelic mutant (T0). -: Non-digested PCR products, +: DdeI-digested PCR products. An undigested band indicates mutation in the NtFT4 gene.
