Oz Mehmet Tufan, Altpeter Angelika, Karan Ratna, Merotto Aldo, Altpeter Fredy
Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS, Gainesville, FL, United States.
DOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL, United States.
Front Genome Ed. 2021 Jul 8;3:673566. doi: 10.3389/fgeed.2021.673566. eCollection 2021.
Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2 = 100 - 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.
甘蔗是全球80%的糖和26%的生物乙醇的来源。然而，其复杂的、高度多倍体的基因组（2n = 100 - 120）阻碍了作物改良。在此，我们报道了甘蔗中高效且可重复的基因靶向（GT），通过可编程核酸酶CRISPR/Cas9诱导的DNA双链断裂的模板介导和同源定向修复（HDR），实现了多个等位基因的精确共编辑。对来自五个独立实验的146株独立转化植株的评估显示，除了分别在25株或18株中出现单一的靶向氨基酸替换W574L或S653I外，在11个株系中出现了靶向核苷酸替换，导致乙酰乳酸合酶（ALS）中同时出现靶向氨基酸替换W574L和S653I。通过对克隆的长聚合酶链反应（PCR）扩增子进行桑格测序，证实了多达三个赋予除草剂耐受性的ALS拷贝/等位基因的共编辑。这项工作将通过靶向核苷酸替换将劣质等位基因转化为优质等位基因，从而实现作物改良。
