基于弹道式转移的瞬时 CRISPR/Cas9 表达可实现小麦的体内基因组编辑。

Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat.

机构信息

Biotechnology Research Laboratories, KANEKA CORPORATION, Takasago, Japan.

Division of Applied Genetics, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 2-1-2 Kannondai, Tsukuba, 305-8602, Japan.

出版信息

Sci Rep. 2018 Sep 26;8(1):14422. doi: 10.1038/s41598-018-32714-6.

Abstract

The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T plants identified plants homozygous for the three homeologous genes, which were all derived from one T plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve in planta genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.

摘要

目前,基因组编辑在作物中的应用仅限于可进行体外培养和再生的品种。在这里,我们报告了一种不需要愈伤组织培养和再生的体内基因组编辑方法。茎尖分生组织(SAM)包含一个表皮下细胞层 L2,在花器官发生过程中,生殖细胞从这里发育而来。通过将表达 CRISPR/Cas9 组件的质粒包裹的金颗粒进行生物弹道传递,靶向 TaGASR7 的成分被轰击到吸胀种子暴露的 SAM 中的胚胎中。选择在 SAM 内显示瞬时 GFP 表达的轰击胚胎,并将其生长为成体植物。通过切割扩增多态性序列分析评估靶基因的突变。在 210 个轰击的植物中,有 11 个(5.2%)携带突变等位基因,其中 3 个(1.4%)的突变在下一代中遗传。T 植物的基因型分析鉴定出了这三个同源基因的纯合子植物,这些植物均来自一个 T 植物。这些植物没有检测到 Cas9 和向导 RNA 基因的整合,表明 CRISPR/Cas9 的瞬时表达引入了突变。总之,我们目前的方法可以在小麦中使用 CRISPR/Cas9 进行体内基因组编辑,并为其他难以编辑的植物物种和变异提供了可能的应用。

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