Figure 1

Construction of all-in-one PtTALEN-ARS plasmids and verification of genome editing activity. (A) Workflow of transgene-free genome editing using all-in-one PtTALEN-ARS plasmids. The plasmids containing CEN/ARS are not inserted into the chromosome and can be removed after mutagenesis. Chr: chromosome. (B) Schematic diagram of all-in-one PtTALEN-ARS plasmids. These plasmids have two antibiotic genes, AmpR and KanR. These are facilitated to construct the all-in-one TALEN-ARS vectors. ProLHC: LHC promoter; terFCP: FCP terminator; L-TALEN: left TALEN; R-TALEN: right TALEN; AmpR: ampicillin resistance gene; ShBle: zeocin resistance gene; KanR: kanamycin resistance gene; ARS: yeast centromere and autonomous replication sequence. (C) Heteroduplex mobility assay (HMA) and Cel-I assay for TALEN target sites of NoNR genes in wild-type and TALEN-induced N. oceanica by the introduction of all-in-one PtTALEN-ARS or TALEN plasmids targeting NoNR. HMA was performed by amplifying the NoNR target site. The Cel-I assay was performed by treating NoNR PCR products with Cel-I enzymes. WT: Wild-type genomic DNAs; ARS-T: total DNAs from all emerged N. oceanica colonies following introduction of all-in-one NoNR PtTALEN-ARS plasmids; T: total DNAs from all emerged N. oceanica colonies following the introduction of all-in-one NoNR PtTALEN plasmids; M: DNA ladder marker.