Lawrenson Tom, Clarke Martha, Kirby Rachel, Forner Macarena, Steuernagel Burkhard, Brown James K M, Harwood Wendy
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
Plant Methods. 2024 Aug 13;20(1):123. doi: 10.1186/s13007-024-01234-y.
CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.
We identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.
Based on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.
CRISPR Cas9和Cas12a是植物系统中报道的两种最常用的可编程核酸酶。现在，这两种酶都有各种各样的组件，其有效性可能不同，对不同物种的适用性也可能不同。我们的目标是开发和优化基于Cas9和Cas12a的系统，用于单子叶植物大麦和小麦的高效基因组编辑，并生产一个用户友好的工具箱，便于谷物群落中的单重和多重编辑。
我们鉴定出一种玉米密码子优化的Cas9，带有13个内含子，并结合由U6和U3启动子驱动的阵列向导，这是大麦中表现最佳的组合，其中100%的T0植株在所有三个靶基因中同时被编辑。当该系统用于小麦时，超过90%的T0植株在所有三个亚基因组靶标中被编辑。对于Cas12a，一种具有8个内含子的拟南芥密码子优化序列与基于tRNA的多向导阵列结合时，在大麦中给出了最佳编辑效率，导致三个同时靶向的基因中有90%的突变等位基因。当我们将这个Cas12a系统应用于小麦时，86%和93%的T0植株在两个同时靶向的基因中发生了突变。我们表明，并非所有内含子插入Cas12a编码序列时对增强诱变的贡献都相同，并且包含多个内含子是有道理的。我们还表明，提高Cas12a诱变效率的两个特征（D156R突变和内含子）的联合效应大于分别应用这些特征的总和。
基于我们的测试结果，我们描述并提供了一种用于大麦和小麦的Cas9和Cas12a的金门模块化克隆系统。该工具箱中经过验证的Cas核酸酶和向导表达盒选项将有助于这两个物种的高效单重和多重诱变。我们在小麦选项中加入了GRF-GIF转化增强盒，以最大限度地提高工作流程效率。
