Panda Debasmita, Karmakar Subhasis, Dash Manaswini, Tripathy Swagat Kumar, Das Priya, Banerjee Sagar, Qi Yiping, Samantaray Sanghamitra, Mohapatra Pradipta Kumar, Baig Mirza J, Molla Kutubuddin A
ICAR National Rice Research Institute, Cuttack, Odisha 753006 India.
Department of Botany, Ravenshaw University, Cuttack, Odisha 753003 India.
aBIOTECH. 2024 Apr 15;5(2):151-168. doi: 10.1007/s42994-024-00139-7. eCollection 2024 Jun.
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, 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, 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.
The online version contains supplementary material available at 10.1007/s42994-024-00139-7.
CRISPR-Cas基因组编辑工具凭借其修改目标基因组位点的简单性和精确性，正在彻底改变农业和基础生物学。软件预测的引导RNA（gRNA）常常无法在目标位点诱导有效的切割。由于复杂的染色质结构，许多目标位点难以接近。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及性。因此，人们花费大量时间和资源用低效的引导物进行编辑实验。尽管体外切割试验可以对gRNA效率进行粗略评估，但它很大程度上排除了天然基因组背景的干扰。瞬时体内测试能在天然基因组背景下对编辑试剂的切割能力进行恰当评估。在此，我们开发了一种改良方案，该方案使用蔗糖梯度从水稻和鹰嘴豆中高效分离原生质体，采用聚乙二醇（PEG）进行转染，并验证CRISPR-Cas9的单向导RNA（sgRNA）切割效率。我们优化了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。我们将含有Cas9和靶向水稻及鹰嘴豆原生质体中基因的sgRNA的质粒载体导入。使用双sgRNA，我们的CRISPR缺失策略通过简单的琼脂糖凝胶电泳就能直接检测基因组编辑是否成功。PCR产物的桑格测序证实了特定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保存长达24/48小时，活力几乎没有损失，这使得在分离和转染之间可以有一个停顿。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（不到7天）验证sgRNA的切割效率。该分离和转染方法还可用于快速验证编辑策略、评估各种编辑试剂、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以及其他应用。
在线版本包含可在10.1007/s42994-024-00139-7获取的补充材料。
