An Effective CRISPR-Cas9 Technology for Efficiently Isolating Transgene-Free Mutants in Arabidopsis, Brassica napus, Strawberry, and Soybean
- 636 Downloads
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 wordsCRISPR-Cas9 GFP Genome editing
- 4.Ding D, Chen K, Chen Y, Li H, Xie K (2018) Engineering introns to express RNA guides for Cas9- and Cpf1-mediated multiplex genome editing. Mol Plant, vol 11, p 542Google Scholar
- 16.Tang T, Yu X, Yang H, Gao Q, Ji H, Wang Y, Yan G, Peng Y, Luo H, Liu K et al (2018) 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. Front Plant Sci 9:1533CrossRefGoogle Scholar