Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
Bo Li,
Chunyang Fu,
Jiawei Zhou,
Fengjiao Hui,
Qiongqiong Wang,
Fuqiu Wang,
Guanying Wang,
Zhongping Xu,
Lianlian Che,
Daojun Yuan,
Yanqin Wang,
Xianlong Zhang,
Shuangxia Jin
Affiliations
Bo Li
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Chunyang Fu
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Jiawei Zhou
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Fengjiao Hui
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Qiongqiong Wang
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Fuqiu Wang
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Guanying Wang
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Zhongping Xu
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Lianlian Che
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Daojun Yuan
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Yanqin Wang
Xinjiang Production and Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Tarim University, Alaer 843300, China
Xianlong Zhang
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Shuangxia Jin
Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1–10 bp in length. The main editing sites are located at positions 11–17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.
