Data_Sheet_1_CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance.pdf

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via 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.

甘蔗贡献了全球80%的食糖产量与26%的生物乙醇产量。然而，其复杂的高度多倍体基因组（2n=100~120）阻碍了作物遗传改良进程。本研究报道了甘蔗中高效且可重复的基因靶向（gene targeting, GT）技术，该技术可通过模板介导及对可编程核酸酶CRISPR/Cas9诱导的DNA双链断裂进行同源定向修复（homology-directed repair, HDR），实现多等位基因的精准共编辑。对5组独立实验获得的146株独立转化植株进行评估后发现：11个株系的乙酰乳酸合酶（acetolactate synthase, ALS）中同时发生了目标氨基酸替换W574L与S653I的靶向核苷酸置换；另有25个株系仅出现W574L替换、18个株系仅出现S653I替换。通过对克隆的长片段聚合酶链式反应（polymerase chain reaction, PCR）扩增子进行桑格测序（Sanger sequencing），证实了可对多达3个赋予除草剂耐受性的ALS拷贝/等位基因进行共编辑。本研究通过靶向核苷酸置换将劣势等位基因转化为优势等位基因，可为甘蔗的作物遗传改良提供技术支撑。