PDF(Pubmed)
Abstract：
BACKGROUND: Base editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants.
RESULTS: In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding.
CONCLUSIONS: Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.
RESULTS: In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding.
CONCLUSIONS: Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.
摘要：
背景：碱基编辑是人工进化创造等位基因多样性并改善农艺性状的强大工具。然而,每个sgRNA靶标的巨大进化潜力被忽视了。并且目前没有高通量方法用于基于大的突变池在单个靶标中产生和表征尽可能多的变化以允许植物中的快速基因定向进化。
结果：在这项研究中，我们建立了一个有效的种系特异性进化系统来筛选拟南芥中有益的等位基因，可用于作物改良。该系统基于强大的卵细胞特异性胞嘧啶碱基编辑器和拟南芥的大种子生产，这使得具有未编辑的野生型等位基因的每个T1植物能够产生数千个独立的T2突变系。它有能力创造广泛的突变系，包括那些含有非典型碱基替换的，以及提供一种节省空间和劳力的方式来存储和筛选产生的突变库。使用这个系统，我们有效地产生抗除草剂的EPSPS，ALS,和可用于作物育种的HPPD变体。
结论：这里，我们证明了碱基编辑介导的人工进化对每个sgRNA靶标的巨大潜力，并设计了一个有效的系统来进行深度进化以利用这一潜力。
结果：在这项研究中，我们建立了一个有效的种系特异性进化系统来筛选拟南芥中有益的等位基因，可用于作物改良。该系统基于强大的卵细胞特异性胞嘧啶碱基编辑器和拟南芥的大种子生产，这使得具有未编辑的野生型等位基因的每个T1植物能够产生数千个独立的T2突变系。它有能力创造广泛的突变系，包括那些含有非典型碱基替换的，以及提供一种节省空间和劳力的方式来存储和筛选产生的突变库。使用这个系统，我们有效地产生抗除草剂的EPSPS，ALS,和可用于作物育种的HPPD变体。
结论：这里，我们证明了碱基编辑介导的人工进化对每个sgRNA靶标的巨大潜力，并设计了一个有效的系统来进行深度进化以利用这一潜力。
