Figure 2

SaCas9 induced mutation in rice.

(a) Expression construct for SaCas9 (upper) and SpCas9 (bottom) in rice. A 3 × FLAG tag and 3 × NLS peptide translationally fused in tandem to the N-terminus of SaCas9. A 1 × FLAG tag was translationally fused to the N-terminus of SpCas9. The OsADH 5′-UTR was added to enhance transcription of the Cas9 gene. OsADH 5′-UTR: 5′ untranslated region of Oryza sativa ALCOHOL DEHYDROGENASE gene. Pea 3A-ter: terminator region of Pea RBCS-3A gene, OsACT1-ter: terminator region of OsACTIN1 gene. (b) CAPS analysis of the OsDL gene locus in SaCas9- and SpCas9-expressing calli. Mutation frequency (%) was calculated from the number of clones with mutation versus total number of sequenced clones indicated under the callus number. -: Non-digested PCR products, +: PstI- or ApaI-digested PCR products. (c) Mutations detected in red rectangle in (b). 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 as 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). (d) Phenotype of dl mutant. Left Wild-type plant, right a regenerated plant from calli with mutation in OsDL gene.