Neelakandan Anjanasree K, Subedi Binita, Traore Sy M, Binagwa Papias, Wright David A, He Guohao
Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA, United States.
Department of Agricultural and Environmental Sciences, Tuskegee University, Tuskegee, AL, United States.
Front Genome Ed. 2022 May 12;4:901444. doi: 10.3389/fgeed.2022.901444. eCollection 2022.
Peanut ( L.), an allotetraploid legume of the Fabaceae family, is able to thrive in tropical and subtropical regions and is considered as a promising oil seed crop worldwide. Increasing the content of oleic acid has become one of the major goals in peanut breeding because of health benefits such as reduced blood cholesterol level, antioxidant properties and industrial benefits such as longer shelf life. Genomic sequencing of peanut has provided evidence of homeologous and genes encoding Fatty Acid Desaturase2 (), which are responsible for catalyzing the conversion of monounsaturated oleic acid into polyunsaturated linoleic acid. Research studies demonstrate that mutations resulting in a frameshift or stop codon in an gene leads to higher oleic acid content in oil. In this study, two expression vectors, pDW3873 and pDW3876, were constructed using Cas9 fused to different deaminases, which were tested as tools to induce point mutations in the promoter and the coding sequences of peanut genes. Both constructs harbor the single nuclease null variant, nCas9 D10A, to which the PmCDA1 cytosine deaminase was fused to the C-terminal (pDW3873) while rAPOBEC1 deaminase and an uracil glycosylase inhibitor (UGI) were fused to the N-terminal and the C-terminal respectively (pDW3876). Three gRNAs were cloned independently into both constructs and the functionality and efficiency were tested at three target sites in the genes. Both constructs displayed base editing activity in which cytosine was replaced by thymine or other bases in the targeted editing window. pDW3873 showed higher efficiency compared to pDW3876 suggesting that the former is a better base editor in peanut. This is an important step forward considering introgression of existing mutations into elite varieties can take up to 15 years making this tool a benefit for peanut breeders, farmers, industry and ultimately for consumers.
花生（Arachis hypogaea L.）是豆科的一种异源四倍体豆科植物，能够在热带和亚热带地区茁壮成长，被认为是全球一种很有前景的油料作物。由于油酸具有降低血液胆固醇水平、抗氧化等健康益处以及延长保质期等工业益处，提高油酸含量已成为花生育种的主要目标之一。花生的基因组测序提供了同源基因和编码脂肪酸去饱和酶2（FAD2）基因的证据，这些基因负责催化单不饱和油酸转化为多不饱和亚油酸。研究表明，FAD2基因中导致移码或终止密码子的突变会使油中的油酸含量更高。在本研究中，构建了两个表达载体pDW3873和pDW3876，它们使用与不同脱氨酶融合的Cas9，作为在花生FAD2基因启动子和编码序列中诱导点突变的工具进行测试。两种构建体都含有单核酸酶无效变体nCas9 D10A，PmCDA1胞嘧啶脱氨酶与C末端融合（pDW3873），而rAPOBEC1脱氨酶和尿嘧啶糖基化酶抑制剂（UGI）分别与N末端和C末端融合（pDW3876）。三个引导RNA（gRNA）被独立克隆到两种构建体中，并在FAD2基因的三个靶位点测试其功能和效率。两种构建体都表现出碱基编辑活性，即在靶向编辑窗口中胞嘧啶被胸腺嘧啶或其他碱基取代。与pDW3876相比，pDW3873显示出更高的效率，表明前者是花生中更好的碱基编辑器。考虑到将现有突变导入优良品种可能需要长达15年的时间，这是向前迈出的重要一步，使该工具对花生育种者、农民、产业乃至消费者都有益处。
