Volume 9, Issue 4, Pages 628-631 (April 2016) Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase  Yongwei Sun, Xin Zhang, Chuanyin Wu, Yubing He, Youzhi Ma, Han Hou, Xiuping Guo, Wenming Du, Yunde Zhao, Lanqin Xia  Molecular Plant  Volume 9, Issue 4, Pages 628-631 (April 2016) DOI: 10.1016/j.molp.2016.01.001 Copyright © 2016 The Author Terms and Conditions

Figure 1 Generation of Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase. (A) A schematic description of the wild-type ALS gene. Note that the locations of target 1, target 2, and the arms are indicated. Target 1 and target 2 are recognized by gRNA1 and gRNA2, respectively. The left and right arms were 100 bp and 46 bp in length, respectively. (B) Linearized construct that was designed to produce Cas9 protein, two gRNAs, and both the donor fragment in the vector and the free donor fragment containing several mutated sites that confer W548L and S627I substitutions. The sequences of mutated target 1, mutated target 2, and the EcoRV site were modified into desired synonymous nucleotides in the donor. (C) PCR products amplified by primers 753F/R were digested with EcoRV. M, DL2000; WT, wild-type. EcoRV cut the PCR products of wild-type, resulting in 488-bp and 265-bp fragments. EcoRV failed to digest the PCR products from eight lines. B99-9 and B99-12 plants appeared to be heterozygous. After EcoRV digestion of the PCR products from B99-9 and B99-12, four fragments (488 bp, 424 bp, 331 bp, and 265 bp) were observed. (D) The sequences of different HDR types. (E) The sequences of two clones from the B99-12 plant. The DSBs were repaired by inserting a DNA fragment that was the reverse complement to the wild-type sequences. Note that the junction sequences are marked with different matching colors. Such insertion would generate two EcoRV fragments (424 bp and 331 bp) (as shown in Figure 1C). The numbers flanking the EcoRV site refer to the distances between the listed sequences. (F) Chromatograms of HDR of the modified ALS gene in the desired region. (G) The plants shown were 36 days after being sprayed with 100 μM bispyribac sodium at the five-leaf stage. The lines on the left side were rice plants with the edited ALS gene and the lines on the right side were wild-type rice plants. Note that wild-type plants died while the edited plants grew normally. For (A, D–F), the gRNA targeting sequences are underlined, and the PAM sequences are boxed. WT ALS refers to the wild-type ALS gene sequence. Differences between the donor sequence and the wild-type ALS gene sequence are shadowed. Molecular Plant 2016 9, 628-631DOI: (10.1016/j.molp.2016.01.001) Copyright © 2016 The Author Terms and Conditions