Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.
Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.
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DOI：
10.1186/s13059-015-0826-7
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发表时间：
2015-11-30
期刊：
影响因子：
12.3
通讯作者：
Harwood W
中科院分区：
文献类型：
--
作者：
Lawrenson T;Shorinola O;Stacey N;Li C;Østergaard L;Patron N;Uauy C;Harwood W
RNA引导的Cas9系统代表了植物基因组编辑的灵活方法。这种方法可以产生敲除或改变靶基因功能的特定突变。它为植物研究提供了宝贵的工具，并为作物改良提供了机会。我们通过靶向多拷贝基因研究了RNA引导的Cas9基因组编辑在大麦（大麦）和甘蓝中的用途和靶特异性要求。在大麦中，我们靶向HvPM 19的两个拷贝，并分别在第一代23%和10%的品系中观察到Cas9诱导的突变。在B。a的靶向导致筛选的10%的第一代植物中的Cas9诱导的突变。此外，表型筛选鉴定具有与靶基因敲除相关的预期矮化表型的T0植物。在大麦和B。将甘蓝稳定的Cas9诱导的突变独立于T-DNA构建体传递至T2植物。我们在两个物种中观察到脱靶活性，尽管在单向导RNA和非靶基因序列之间存在至少一个错配。在大麦中，无转基因植物在HvPM 19的靶拷贝和非靶拷贝中具有并发突变。我们证明了使用RNA引导的Cas9在大麦和B的靶基因中产生突变。并且显示这些突变的稳定传递，从而建立了在这些物种中快速表征基因功能的潜力。此外，所报告的脱靶效应为针对作物多基因家族成员提供了潜在的困难和具体的机会。本文的在线版本（doi：10.1186/s13059-015-0826-7）包含补充材料，可供授权用户使用。
The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement. We investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19. We demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops. The online version of this article (doi:10.1186/s13059-015-0826-7) contains supplementary material, which is available to authorized users.