Figure 5.

Identification of CRISPR/Cas9-induced mutation in the GmNARK (Rj7) target loci in soybean with AtGCSpro ₂₄₁₁ and 2×35S to drive Cas9, respectively. The sequence of an sgRNA designed to target a site within the first exon region of Rj7. The protospacer-adjacent motif (PAM) sequence is highlighted in blue and the EcoRI restriction site is underlined (A). PCR-RE assays to detect CRISPR/Cas9-induced mutation in the Rj7 target loci from 30 different independent p2×35Spro-Cas9 hairy roots (B). Genotypes of eight representative mutants from transformed with p2×35Spro-Cas9 hairy roots identified by sequencing (C). PCR-RE assays to detect CRISPR/Cas9-induced mutation in the Rj7 target loci from 30 different independent pAtGCSpro ₂₄₁₁-Cas9 hairy roots (D). Genotypes of six representative rj7 mutants from transformed with pAtGCSpro ₂₄₁₁-Cas9 hairy roots identified by sequencing (E). In sections B and D, Lanes WT and WTE, undigested PCR amplification fragment and digested wild-type controls by EcoRI, respectively. Lanes 1–30, different independent transgenic hairy roots. In sections C and E, deletions and insertions are indicated as dashes and blue letter, respectively. The types and number(s) of indels are indicated in the right column. Examples given of mutation at target site in the p2×35Spro-Cas9 and pAtGCSpro ₂₄₁₁-Cas9 hairy root, respectively (F). Black arrows indicate the site of indels mutation. The PAM regions and mutated target sites are shown in the black box.