An Effective CRISPR-Cas9 Technology for Efficiently Isolating Transgene-Free Mutants in Arabidopsis, Brassica napus, Strawberry, and Soybean

Part of the Springer Protocols Handbooks book series (SPH)


The CRISPR-Cas9 technique is a highly valuable tool in creating new materials for both basic and applied researches. Here, we inserted a fluorescent tag (sGFP) into the CRISPR-Cas9 vector pKSE401 to facilitate a visual screening of mutants. This modified vector was named pKSE401G and tested in several dicot plant species, including Arabidopsis, Brassica napus, Fragaria vesca (strawberry), and Glycine max (soybean). Consequently, the candidate mutants were isolated through fluorescence screening, and the transgene-free mutants were sufficiently identified in Arabidopsis and B. napus in the next generation based on the absence of GFP fluorescence. 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.

Key words

CRISPR-Cas9 GFP Genome editing 


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina
  2. 2.Key Laboratory of Horticultural Plant Biology (Ministry of Education)College of Horticulture and Forestry Sciences, Huazhong Agricultural UniversityWuhanChina

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