Volume 8, Issue 12, Pages 1820-1823 (December 2015) High-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR/Cas9 System  Liuhua Yan, Shaowei Wei, Yaorong Wu, Ruolan Hu, Hongju Li, Weicai Yang, Qi Xie  Molecular Plant  Volume 8, Issue 12, Pages 1820-1823 (December 2015) DOI: 10.1016/j.molp.2015.10.004 Copyright © 2015 The Author Terms and Conditions

Figure 1 pYAO:hSpCas9 Edited Arabidopsis Genes More Efficiently than 35S:hSpCas9 through Floral Dip Transformation. (A) Structure of the CRISPR/Cas9 binary vectors for Arabidopsis transformation by floral dip. The hSpCas9 cassette is driven by the 35S (left) or YAO (right) promoter, while sgRNA is controlled by the AtU6-26 promoter. NLS, nuclear localization sequence. (B) Statistical results of phenotypes and mutations in T1 transgenic plants of Arabidopsis. (C) Phenotypes of T1 and T2 transgenic plants of the BRI1 sgRNA target. 35S:hSpCas9-BRI1-sgRNA T1 transgenic plants line 6 (35S-6 T1) and line 18 (35S-18 T1) exhibited a small-seedling phenotype compared with the wild-type Col-0. pYAO:hSpCas9-BRI1-sgRNA T1 transgenic plants lines 3 and 4 (pYAO-3 T1 and pYAO-4 T1) showed rolling leaves and retarded growth phenotypes, similarly to a bri1 mutant. 35S-6 T2, 35S-18 T2, pYAO-3 T2, and pYAO-4 T2 showed three types of phenotype including dwarf and rolling leaves, like bri1, mosaics, and wild-type. The T1 plants were screened on 1/2 MS plates with hygromycin and carbenicillin for 15 days and transplanted in soil for 10 days before photographing. The T2 plants were sown on 1/2 MS medium for 8 days before photographing. Scale bars, 1 cm. (D) RFLP analysis of genomic DNA from all of the T1 plants using the PCR primer pair P1 (5′-GATGGGATGAAGAAAGAGTG-3′) and P2 (5′-CTCATCTCTCTACCAACAAG-3′). EcoRV was used to digest the PCR products. M, DNA marker. (E) DNA sequencing peaks showed evidence of successful gene editing in the target region of BRI1. More peaks represent more mutant alleles. (F) Representative sequences of single mutant alleles of BRI1 identified from the 35S T1-6 (35S:hSpCas9-BRI1-sgRNA T1 transgenic line 6) and the pYAO T1-16 (pYAO:hSpCas9-BRI1-sgRNA T1 transgenic line 16). (G) Representative sequences of several mutant alleles of BRI1 identified from the pYAO:hSpCas9-BRI1-sgRNA T1 transgenic plant line 3. The wild-type (WT) sequence is shown at the top of (F) and (G) with the PAM sequence highlighted in red. The target sequence is in the frame. Molecular Plant 2015 8, 1820-1823DOI: (10.1016/j.molp.2015.10.004) Copyright © 2015 The Author Terms and Conditions