Introduction of a second "Green Revolution" mutation into wheat via in planta CRISPR/Cas9 delivery

Abstract

Direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem via particle bombardment enabled introduction of a semidwarf1-orthologous mutation into an elite wheat variety.

Figure 1

In planta RNP-mediated genome editing in wheat. a, The workflow of the iPB-RNP method utilizing wheat SAMs. b, CAPS analysis of E₀ plants at the TaQsd1 locus. The PCR products were amplified by an A, B, and D genome common primer set (Supplemental Table S1). WT, undigested PCR products; WT/+, Pst I digested PCR products. Red and blue arrows indicate undigested and digested bands after Pst I treatment, respectively. A 100-bp ladder was used as a size marker. c, The genotypes of Q1, Q5, Q7, Q8, and Q9 plants as identified by sequencing. The black and red characters indicate the gRNA and PAM sequences, respectively. The Pst I restriction site is underlined. Inserted nucleotides are shown in green characters. d, Summary of genome editing experiment on locus sites of TaQsd1, TaOr_t0, TaOr_t1, and TaHRGP-like1_t2 using the iPB-RNP method.

Figure 2

Introduction of sd1 mutations in wheat. a, Target sequences conserved among the three homoeologous TaSD1 genes were selected using the CRISPRdirect software. The locations of the target sequences are indicated by arrows. The boxes and lines indicate exons and introns, respectively. The three exons in TaSD1 are numbered. b, Summary of the CAPS analysis of bombarded E₀ plants and their progeny. c, CAPS assays of selected positive E₀ plants using genome-specific primers. WT/D, WT fragment digested with Sal I; WT/U, WT fragment undigested. Red and blue arrows indicate undigested and digested bands after Pst I treatment, respectively. d, Mutations detected within the target region of positive E₀ plants. The gRNA sequence is underlined in the WT sequence. Protospacer adjacent motif (PAM) sequences are indicated in blue letters. Insertions and deletions are indicated in red letters. e, A genome-specific CAPS assay of a tasd1 mutant line (H7-1, E₁). Red and blue arrows indicate undigested and digested bands after Sal I treatment, respectively. The A, B, and D genome sequences of H7-1 are aligned with the A genome sequence of the WT. The inserted nucleotide and PAM sequence are indicated by red and blue letters, respectively. f, Comparison of plant stature of tasd1 (left) and WT (right) plants. Average tiller height based on measurements of all plants. Data represent the mean ± se of seven tasd1 and six WT plants. Asterisks indicate statistically significant difference (t test, P < 0.01). g, Comparison of grain yield. Average total grain numbers and average total grain weight for each plant are shown. The data represent the mean ± se of seven tasd1 and six WT plants.