Expanding the Range of CRISPR/Cas9 Genome Editing in Rice Xixun Hu, Chun Wang, Yaping Fu, Qing Liu, Xiaozhen Jiao, Kejian Wang  Molecular Plant  Volume 9, Issue 6, Pages 943-945 (June 2016) DOI: 10.1016/j.molp.2016.03.003 Copyright © 2016 The Author Terms and Conditions

Figure 1 Genome Editing in Rice Using the VQR and VRER Cas9 Variants. (A) Schematic illustration of the generation of VQR and VRER. NLSs, nuclear localization signals. (B) PCR/RE assay of mutations around NAL1-Q2. Its PCR products were digested with PstI. Lane one is DNA markers; the last two lanes are wild-type controls digested and undigested with PstI, respectively; lines 3, 14, 15, and 18 are putative monoallelic mutants; line 19 is a putative biallelic mutant. The arrowhead indicates the bands used for identifying mutations. (C) Sequencing assay of mutations around NAL1-Q2. Line 18 is a chimera (more than one mutational type); line 19 is a biallelic homozygous mutant; the others are monoallelic mutants. The top sequence comes from the wild-type with the target (blue) and corresponding PAMs (red). A PstI site is underlined. (D) Phenotypes of the nal1 mutant. Upper is wild-type. Below is the nal1 mutant, which has a typical narrow leaf phenotype. Bar, 1 cm. (E) Mutation analyses at the target sites of the VQR variant. The biallelic mutations include the biallelic homozygous mutant and the biallelic heterozygous mutant. (F) Phenotypes of the lg1 and gl1-1 double mutant generated by VQR. Upper is wild-type. Below is the lg1 and gl1-1 double mutant. The double mutant exhibited the phenotypes of the loss of cuticular wax (indicated by red arrows), laminar joint, auricle, and ligule (indicated by yellow arrows). Bar, 1 cm. (G) Representation of the number of potentially targetable sites in the rice genome by the VQR variant, the VRER variant, and wild-type Cas9. Molecular Plant 2016 9, 943-945DOI: (10.1016/j.molp.2016.03.003) Copyright © 2016 The Author Terms and Conditions