Milner Matthew J, Sharma Manisha, Bates Ruth E, Whiting Michelle, Craze Melanie S, Miller Peter, Brooks Jack, Kouidri Allan, Wallington Emma J
NIAB, Cambridge, United Kingdom.
Front Plant Sci. 2024 Dec 5;15:1426184. doi: 10.3389/fpls.2024.1426184. eCollection 2024.
Cereal transformation and gene editing can be a complex and costly undertaking. It is therefore important to validate and understand the performance of the components to achieve high rates of transformation and gene editing. Here, we have made a direct comparison of different CRISPR/Cas9 guide systems to target the genome in three cereal species. We show that the guide sequences driven by the same pol II promoter in rice, wheat and barley show large differences in editing efficiency. The differences seen were based on the way the guides were presented and factors outside of the guide sequence itself. While both the tRNA system and ribozyme system performed well in rice, their effectiveness varied in wheat and barley. Specifically, the tRNA system outperformed the ribozyme system, achieving higher rates of editing in stable transformed plants. Overall, high levels of editing are observed in all three species when strong expression of the SpCas9 is coupled with the CmYLCV promoter to drive a tRNA array of guide RNAs. Stable inheritance is also achievable in all three species when plants are sampled shortly after the tissue culture concludes. Overall, inheritance rates were above 85% in all three species, particularly when mutations are detected early after plants emerge from tissue culture.
谷物转化和基因编辑可能是一项复杂且成本高昂的工作。因此，验证和了解各组件的性能对于实现高转化率和基因编辑率很重要。在此，我们对三种谷物物种中靶向基因组的不同CRISPR/Cas9引导系统进行了直接比较。我们发现，由水稻、小麦和大麦中相同的聚合酶II启动子驱动的引导序列在编辑效率上存在很大差异。观察到的差异基于引导序列的呈现方式以及引导序列本身之外的因素。虽然tRNA系统和核酶系统在水稻中均表现良好，但它们在小麦和大麦中的有效性有所不同。具体而言，tRNA系统优于核酶系统，在稳定转化的植株中实现了更高的编辑率。总体而言，当SpCas9的强表达与CmYLCV启动子相结合以驱动引导RNA的tRNA阵列时，在所有三个物种中均观察到高水平的编辑。当组织培养结束后不久对植株进行取样时，所有三个物种也都能实现稳定遗传。总体而言，所有三个物种的遗传率均高于85%，尤其是在植株从组织培养中长出后早期检测到突变时。
