Generation of conditional knockout mutants of an essential gene.

(A) Gene model of the MpMPK1 gene and the gRNA target site. Grey boxes and lines indicate exons and introns. “ATG” and “Stop” denote the predicated initiation and termination codons. PAM, target, and intron sequences are shown in blue, in bold, and by small letters, respectively. Note that the third base (g) of the PAM sequence is in intron 1 and that the first base of exon 2 is not G. (B) Targeted mutagenesis rate of the endogenous MpMPK1 locus by CRISPR/Cas9. Bar graphs show proportions of clonal frameshift (magenta), clonal in-frame (green), mosaic (yellow), and no mutations (grey) that were identified in plants transformed with pMpGE010 containing the gRNA shown in (A) only (Exp. 1) or together with MpMPK1 cDNA-containing pMpGWB337 (Exp. 2) or pMpGWB337tdTN (Exp. 3). (C) Target-site sequences of plants obtained in Exp. 3. Sequences of wild type (WT) and transgenic lines (#1 to #8 except #4) are shown as in (A). Inserted or deleted bases are colored in magenta with their numbers in parentheses. In line #4, a 2 bp insertion and a large 970 bp deletion that covers the predicted initiation codon were identified (magenta). (D) Growth defect manifested by conditional deletion of the MpMPK1 cDNA. Gemmae of transgenic line #4 were (+) or were not (–) subjected to heat shock (HS) and dexamethasone (DEX) treatment on day 0 and day 1 and grown at 22°C for 2 weeks (top) before observation by fluorescent microscopy for tdTomato-NLS (bottom). Scale bars = 5 mm (top); 1 mm (bottom). (E) Confirmation of deletion events by genomic PCR. From HS/DEX-treated plants (line #4), tdTomato-positive, disorganized tissues ("KO") and tdTomato-negative, thallus-looking sectors [such as the arrow in (D); "non"] were collected from two different individuals and analyzed by genomic PCR using primer sets 1 and 2 shown in the schematic illustration of the construct used (predicted product sizes are indicated) and primer set 3 shown in panel (C).