Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants
He Yubing,
Zhu Min,
Wang Lihao,
Wu Junhua,
Wang Qiaoyan,
Wang Rongchen,
Zhao Yunde
Affiliations
He Yubing
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070, China
Zhu Min
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Wang Lihao
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Wu Junhua
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Wang Qiaoyan
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Wang Rongchen
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
Zhao Yunde
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116, USA; Corresponding author.
Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice. Keywords: genome editing, suicide gene, transgene killer CRISPR, Cas9, transgene-free
