Efficient Editing of <i>S</i><i>oCSLD2</i> by CRISPR/Cas9 Affects Morphogenesis of Root Hair in Spinach
Yingping Cao,
Yue Xu,
Yue Zhang,
Heng Zhang,
Zhi Qin,
Chen Bai,
Hailing Zhang,
Dongmei Ma,
Quanhua Wang,
Chunxiang Fu,
Meihong Sun,
Shaojun Dai
Affiliations
Yingping Cao
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Yue Xu
Shandong Technology Innovation Center of Synthetic Biology, Shandong Provincial Key Laboratory of Energy Genetics and CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266000, China
Yue Zhang
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Heng Zhang
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Zhi Qin
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Chen Bai
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Hailing Zhang
Grass and Science Institute of Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
Dongmei Ma
Breeding Base for State Key Laboratory of Land Degradation and Ecological Restoration in Northwestern, Key Laboratory of Restoration and Reconstruction of Degraded Ecosystems in Northwestern China of Ministry of Education, School of Ecology and Environment, Ningxia University, Yinchuan 750021, China
Quanhua Wang
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Chunxiang Fu
Shandong Technology Innovation Center of Synthetic Biology, Shandong Provincial Key Laboratory of Energy Genetics and CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266000, China
Meihong Sun
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Shaojun Dai
Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we established a CRISPR/Cas9-based gene-editing system for spinach hairy roots and edited two cellulose synthase-like D (CSLD) genes (SoCSLD2 and SoCSLD3) that were involved in root-hair formation of spinach hairy roots. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the replacement accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutants of SoCSLD2 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic mutant lines displayed bulking and short root hairs, which resembled the characteristics of Arabidopsis atcsld2 mutants. The transcriptomic analysis further revealed that multiple gene expressions for cell-wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in socsld2 mutants. This indicates that SoCSLD2 was successfully knocked out in spinach root hairs using the CRISPR/Cas9-based gene editing system.
