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

Abstract

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