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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  Brassica napus  using an available CRISPR/Cas9 vector  pKSE401 , while isolation of Cas9-free mutants is laborious and inefficient. Here, we inserted a fluorescence tag (sGFP) driven by the constitutive  35S  promoter into  pKSE401  to facilitate a visual screen of mutants. This modified vector was named  pKSE401G  and tested in several dicot plant species, including  Arabidopsis ,  B. napus ,  Fragaria vesca  (strawberry), and  Glycine max  (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  Arabidopsis  and  B. napus  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  Arabidopsis  in the T2 generation and  B. napus  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,  pKSE401G  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.

frontiers in plant science

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