PDF(Pubmed)
Abstract：
The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00139-7.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00139-7.
摘要：
CRISPR-Cas基因组编辑工具以其简单和精确的修饰靶基因组基因座的能力正在彻底改变农业和基础生物学。软件预测的指导RNA(gRNA)通常不能在靶基因座处诱导有效切割。由于复杂的染色质结构,许多靶基因座是不可接近的。目前,没有合适的工具可用于预测基因组靶位点的结构及其可达性。因此，大量的时间和资源花费在执行编辑实验与低效的指南。尽管体外切割测定可以提供gRNA效率的粗略评估，它在很大程度上排除了天然基因组背景的干扰。瞬时体内测试给出了在天然基因组背景下编辑试剂的切割能力的适当评估。这里,我们开发了一种改进的协议，提供高效的原生质体分离水稻，拟南芥,还有鹰嘴豆,使用蔗糖梯度，使用PEG（聚乙二醇）转染，和验证CRISPR-Cas9的单指导RNA(sgRNA)切割效率。我们已经优化了PEG介导的原生质体转染的各种参数，并使用我们的方案在单子叶植物和双子叶植物中均实现了高转染效率。我们在水稻中引入了含有Cas9和sgRNA靶向基因的质粒载体，拟南芥,和鹰嘴豆原生质体。使用双重sgRNA，我们的CRISPR缺失策略可通过简单的琼脂糖凝胶电泳直接检测基因组编辑的成功.PCR产物的Sanger测序证实了特定sgRNA的编辑效率。值得注意的是,我们证明，分离的原生质体可以储存长达24/48小时，几乎没有活力损失，允许分离和转染之间的暂停。这种用于原生质体分离和转染的高效方案能够在进行稳定转化之前快速(少于7天)验证sgRNA切割效率。分离和转染方法也可用于编辑策略的快速验证，评估不同的编辑试剂，从转染的原生质体再生植物，基因表达研究，蛋白质定位和功能分析，和其他应用。
■在线版本包含补充材料，可在10.1007/s42994-024-00139-7获得。
■在线版本包含补充材料，可在10.1007/s42994-024-00139-7获得。
