Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.
Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.
复制标题
DOI：
10.1007/978-1-4939-8944-7_14
复制
发表时间：
2018-11
影响因子：
--
通讯作者：
T. Lawrenson;W. Harwood
中科院分区：
文献类型：
--
作者：
T. Lawrenson;W. Harwood
与拟南芥模型相比，基因敲除突变体是一种宝贵的反向遗传学工具，但在作物物种中尚未得到很好的发展。然而，CRISPR/Cas9的出现改变了这种情况，使得包括大麦在内的许多作物都可以产生这种突变体。单个T-DNA构建体可以转化为大麦未成熟胚和通过组织培养再生的含有靶向突变的稳定转基因株系。突变在T0植株中被检测到，并在随后的T1和T2代中继续与T-DNA分离，留下非转基因的系，并在目标位点携带各种突变。这些突变可以针对感兴趣的特定基因，以导致功能丧失，从而产生敲除突变体。
Knockout mutants are an invaluable reverse genetics tool which have not been well developed in crop species compared to models like Arabidopsis. However, the emergence of CRISPR/Cas9 has changed this situation making the generation of such mutants accessible to many crops including barley. A single T-DNA construct can be transformed into barley immature embryos and stable transgenic lines regenerated through tissue culture which contain targeted mutations. Mutations are detected in T0 plants and go on in subsequent T1 and T2 generations to segregate from T-DNA, leaving lines which are non-transgenic and carrying a variety of mutations at the target locus. These mutations can be targeted to a particular gene of interest in order to bring about a loss of function creating a knockout mutant.