Present address: CSIRO, Agriculture and Food, Canberra, ACT, Australia.
Present address: New South Wales Department of Primary Industries, Research Excellence, Orange, NSW, Australia.
BMC Biotechnol. 2019 Oct 30;19(1):71. doi: 10.1186/s12896-019-0565-z.
The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.
The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.
We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.
CRISPR-Cas9 系统是作物基因组编辑的强大而多功能的工具。然而，在多倍体物种中实现高效和特异性编辑可能是一个挑战。CRISPR-Cas9 系统的效率和特异性取决于所使用的 gRNA。在这里，我们评估了针对六倍体小麦原生质体中 5-烯醇丙酮酰莽草酸-3-磷酸合酶 (EPSPS) 的七种 gRNA 的活性和特异性。EPSPS 是广泛使用的除草剂草甘膦的生物靶标。
七种 gRNA 在其靶标活性上有很大差异，平均插入缺失频率范围从 0%到约 20%。实验测定的和计算机预测的靶标 gRNA 活性之间没有明显的相关性。在引导序列的种子区域内存在单个错配大大降低但没有完全消除 gRNA 活性，而存在额外的错配或没有 PAM 则完全消除 gRNA 活性。高达 8.5%的总插入缺失频率检测到 DNA 载体衍生序列的大插入（≥20bp）。其中一种 gRNA 表现出几种特性，使其有可能开发出非转基因草甘膦抗性小麦。
我们已经建立了一种在六倍体小麦原生质体中验证 gRNA 的快速可靠方法。该方法可用于鉴定具有有利特性的 gRNA。我们的方法特别适用于多倍体物种，但应该适用于任何可进行原生质体转化的植物物种。
