柳枝稷木质素基因F5H的高效编辑

柳枝稷(Panicum virgatum)是重要的C4多年生木质纤维素类生态能饲草。为了快速创制细胞壁转化效率高的能饲草新资源,以异源四倍体柳枝稷品种Alamo为材料,克隆了其木质素合成途径的阿魏酸-5-羟基化酶基因PvF5H,并根据其序列设计编辑靶点,用于构建CRISPR/Cas9-PvF5H编辑载体,最后通过农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法,获得了59株柳枝稷转基因阳性植株。测序分析表明,PvF5H在94.9%的转基因植株中被编辑,纯合编辑效率为55.4%。该研究建立了高效的柳枝稷基因编辑系统,实现了对细胞壁品质相关靶基因的有效编辑,为今后能饲草新品种的培育奠定了基础。

Anthocyanin-assisted Agrobacterium infiltration for the rapid evaluation of genome editing efficiencies across multiple plant species

CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing

Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.

Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1)to nick the target DNA strand

The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro cleavage assays and base editing analysis,we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1).We also show that these nicks are made on the target DNA strand.These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets.This system provides a novel tool for achieving trait stacking in plants.

CRISPR/Cas9-introduced single and multiple mutagenesis in strawberry

CRISPR/Cas9-mediated genome editing is a powerful tool for life science research. Recently, strawberry (Fragaria × ananassa), an important horticultural crop, has emerged as a model organism for investigating the regulatory mechanisms of fruit development and ripening (Shulaev et al., 2011; Jia et al., 2013, 2017; Kang et al., 2013; Han et al., 2015). While most cultivated strawberries