Developing an efficient CRISPR/Cas9 system in plants: a test case in Lotus japonicus

CRISPR/Cas9-based genome editing is usually employed for both plant functional genomics and trait engineering, with associated components being most commonly delivered via Agrobacterium-mediated T-DNA transformation. Establishing a functional CRISPR/Cas9 system, however, remains an arduous task, despite the well-established delivery method. Here, leveraging the principles of synthetic biology and the Design-Build-Test-Learn framework (DBTL), we describe a step-by-step guide for developing an efficient T-DNA-based CRISPR/Cas9 system in plants, using Lotus japonicus as a case study. The first step involves rational design of the T-DNA region by selecting characterized genetic parts and arranging expression cassettes logically, in various combinations, generating multiple T-DNA architecture variants. The second step includes the assembly of the T-DNA variants into constructs via a flexible cloning scheme. The third and fourth steps include introducing the T-DNA variants into the plant and evaluating their mutation efficiency, using both an optimized T7 endonuclease I-based assay and an amplicon sequencing analysis. We employed two iterative DBTL cycles to identify and improve sub-optimal parts in our constructs and investigated factors that may affect the mutation efficiency of the system. We also observed that the CRISPR/Cas9 activity, but not specificity, can be affected by both the codon-optimization of the Cas9 sequence and the plant growth conditions.This study provides a reference for establishing an efficient CRISPR/Cas9 system in plants by outlining key methodological considerations for successful implementation.

基于CRISPR/Cas9的基因组编辑技术（CRISPR/Cas9-based genome editing）通常应用于植物功能基因组学与性状工程研究，其相关组分最常通过农杆菌介导的T-DNA转化进行递送。尽管该递送方法已较为成熟，但构建一套功能性CRISPR/Cas9系统仍是一项艰巨的任务。本研究依托合成生物学（synthetic biology）原理与设计-构建-测试-学习框架（Design-Build-Test-Learn framework, DBTL），以百脉根（Lotus japonicus）为模式物种，详细阐述了一套构建高效T-DNA型植物CRISPR/Cas9系统的分步指南。第一步涉及T-DNA区域的理性设计：通过筛选已表征的遗传元件，并以多种组合形式逻辑排布表达盒，从而生成多种T-DNA结构变体；第二步为通过灵活克隆方案将各类T-DNA变体组装至表达载体中；第三步与第四步分别为将T-DNA变体导入植物宿主，以及利用优化后的T7核酸内切酶I检测体系与扩增子测序分析，评估其基因突变效率。本研究通过两轮迭代的DBTL循环，对载体中亚最优的元件开展鉴定与优化，并探究了诸多可能影响该系统基因突变效率的因素。此外，本研究还发现，CRISPR/Cas9的活性（而非特异性）可同时受Cas9序列的密码子优化与植物生长条件的调控。本研究通过梳理成功构建该系统的关键方法学要点，为在植物中建立高效CRISPR/Cas9系统提供了重要参考依据。