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A gene-editing/complementation strategy for tissue-specific lignin reduction while preserving biomass yield
Biotechnology for Biofuels ( IF 6.3 ) Pub Date : 2021-09-03 , DOI: 10.1186/s13068-021-02026-5
Hasi Yu 1, 2 , Chang Liu 1 , Richard A Dixon 1, 2
Affiliation  

Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR–Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene’s open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, and are stable over at least four generations. The method provides a rapid approach for generating reduced lignin trees or crops with one single transformation event, and, paired with a range of tissue-specific promoters, provides a general strategy for optimizing loss-of-function traits that are associated with growth penalties. This method should be applicable to any plant species in which transformation and gene editing are feasible and validated vessel-specific promoters are available.

中文翻译:

一种减少组织特异性木质素同时保持生物质产量的基因编辑/互补策略

次生细胞壁的木质化是导致木质纤维素生物质难以分解燃料和化学品的主要因素。基因改造可以降低木质素含量并提高糖化效率,但通常以中度至重度生长惩罚为代价。我们开发了一种方法,使用单个 DNA 构建体,该构建体使用 CRISPR-Cas9 基因编辑来敲除木质素单体生物合成的内源基因的表达,同时表达该基因的开放阅读框的修改版本,从而避免被切割。 Cas9 系统并以组织特异性方式补充引入的突变。在容器中表达互补的开放阅读框允许拟南芥植物再生木质素减少,野生型生物量产量,每株植物的细胞壁糖产量提高四倍。上述表型在纯合子和双等位基因杂合子 T1 品系中均可见,并且在至少四代中是稳定的。该方法提供了一种通过单个转化事件生成木质素减少的树木或作物的快速方法,并且与一系列组织特异性启动子配对,提供了一种优化与生长惩罚相关的功能丧失性状的通用策略。这种方法应该适用于任何可以进行转化和基因编辑并且经过验证的容器特异性启动子可用的植物物种。该方法提供了一种通过单个转化事件生成木质素减少的树木或作物的快速方法,并且与一系列组织特异性启动子配对,提供了一种优化与生长惩罚相关的功能丧失性状的通用策略。这种方法应该适用于任何可以进行转化和基因编辑并且经过验证的容器特异性启动子可用的植物物种。该方法提供了一种通过单个转化事件生成木质素减少的树木或作物的快速方法,并且与一系列组织特异性启动子配对,提供了一种优化与生长惩罚相关的功能丧失性状的通用策略。这种方法应该适用于任何可以进行转化和基因编辑并且经过验证的容器特异性启动子可用的植物物种。
更新日期:2021-09-03
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