Citrus Research and Education Center, University of Florida, Lake Alfred, Florida, USA.
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
BMC Biotechnol. 2020 Nov 10;20(1):58. doi: 10.1186/s12896-020-00652-9.
Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study.
Citrus sinensis 'EV2' embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6-26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1-2 bp), substitution (1 bp), or deletion (1-3 bp) mutations that occurred close to the protospacer adjacent motif.
Both the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.
开发精确的基因组编辑策略是生产可用于研究基因功能和帮助理解品种遗传特性的编辑植物的前提。柑橘胚胎细胞培养可用于快速产生大量基因组编辑的转化柑橘系。本研究评估了使用两个构建体（pC-PDS1 和 pC-PDS2）在这些细胞的基因组中引入特定突变的能力。
柑橘‘EV2’胚胎细胞培养物适合农杆菌介导的基于 CRISPR/Cas9 的基因组编辑。针对脱植烯酶（PDS）基因两个位置的向导 RNA（gRNA）分别由拟南芥 U6-26 启动子（pC-PDS1）驱动或在 CmYLCV 启动子（pC-PDS2）的控制下组装为 Csy4 阵列。所有转基因胚胎均为完全白化，未观察到斑驳表型。本研究评估了每个构建体的 12 个品系，大多数品系含有靠近原间隔基序的插入（1-2 bp）、替换（1 bp）或缺失（1-3 bp）突变。
pC-PDS1 和 pC-PDS2 均可成功编辑柑橘胚胎细胞培养物。然而，编辑效率取决于 gRNA，这证实了选择适当的 gRNA 对于使用 CRISPR/Cas9 技术成功进行基因组编辑至关重要。此外，胚胎细胞培养物的利用为柑橘的成功基因组编辑提供了另一种选择。
