AUTHOR=Tang Ting , Yu Xiwen , Yang Hong , Gao Qi , Ji Hongtao , Wang Yanxu , Yan Guanbo , Peng Yan , Luo Huifeng , Liu Kede , Li Xia , Ma Chaozhi , Kang Chunying , Dai Cheng 

TITLE=Development and Validation of an Effective CRISPR/Cas9 Vector for Efficiently Isolating Positive Transformants and Transgene-Free Mutants in a Wide Range of Plant Species

JOURNAL=Frontiers in Plant Science

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2018.01533

DOI=10.3389/fpls.2018.01533

ISSN=1664-462X

ABSTRACT=<p>The CRISPR/Cas9 technique is a highly valuable tool in creating new materials for both basic and applied researches. Previously, we succeeded in effectively generating mutations in <italic>Brassica napus</italic> using an available CRISPR/Cas9 vector <italic>pKSE401</italic>, while isolation of Cas9-free mutants is laborious and inefficient. Here, we inserted a fluorescence tag (sGFP) driven by the constitutive <italic>35S</italic> promoter into <italic>pKSE401</italic> to facilitate a visual screen of mutants. This modified vector was named <italic>pKSE401G</italic> and tested in several dicot plant species, including <italic>Arabidopsis</italic>, <italic>B. napus</italic>, <italic>Fragaria vesca</italic> (strawberry), and <italic>Glycine max</italic> (soybean). Consequently, GFP-positive plants were readily identified through fluorescence screening in all of these species. Among these GFP-positive plants, the average mutation frequency ranged from 20.4 to 52.5% in <italic>Arabidopsis</italic> and <italic>B. napus</italic> with stable transformation, and was 90.0% in strawberry and 75.0% in soybean with transient transformation, indicating that the editing efficiency resembles that of the original vector. Moreover, transgene-free mutants were sufficiently identified in <italic>Arabidopsis</italic> in the T2 generation and <italic>B. napus</italic> in the T1 generation based on the absence of GFP fluorescence, and these mutants were stably transmissible to next generation without newly induced mutations. Collectively, <italic>pKSE401G</italic> provides us an effective tool to readily identify positive primary transformants and transgene-free mutants in later generations in a wide range of dicot plant species.</p>