Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing
Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing
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单转录单位 CRISPR 2.0 系统，用于强大的 Cas9 和 Cas12a 介导的植物基因组编辑
DOI：
10.1111/pbi.13068
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发表时间：
2019-07-01
影响因子：
13.8
通讯作者：
Zhang, Yong
中科院分区：
文献类型：
--
作者：
Tang, Xu;Ren, Qiurong;Zhang, Yong
CRISPR-Cas9 和 Cas12a 是两种强大的基因组编辑系统。 CRISPR在植物中的表达通常通过混合双启动子系统来实现，其中Cas蛋白由Pol II启动子表达，指导RNA由物种特异性Pol III启动子（例如U6或U3）表达。为了实现协调表达和紧凑的载体包装，需要在单个 Pol II 启动子下表达两个 CRISPR 组件。此前，我们展示了第一代单转录单元（STU）-Cas9系统STU-Cas9-RZ，该系统基于锤头核酶，用于处理单引导RNA（sgRNA）。在这项研究中，我们开发了两种新的STU-Cas9系统和一种STU-Cas12a系统用于植物应用，统称为STU CRISPR 2.0系统。我们展示了这些用于水稻基因组编辑的系统，具有瞬时表达和稳定转基因。两个 STU-Cas9 2.0 系统分别使用 Csy4 核糖核酸酶和内源 tRNA 处理系统处理 sgRNA。 STU-Cas9-Csy4 和 STU-Cas9-tRNA 系统都比我们的第一代 STU-Cas9-RZ 系统和传统的混合双启动子系统表现出更强大的基因组编辑效率。我们进一步应用 STU-Cas9-tRNA 系统来比较两种基于 rAPOBEC1 和 PmCDA1 胞苷脱氨酶的 C 到 T 碱基编辑系统。结果表明基于 STU 的 PmCDA1 碱基编辑系统在水稻中非常有效。 STU-Cas12a 系统基于 Cas12a 对 CRISPR RNA (crRNA) 阵列的自我处理，也被开发并演示用于表达单个 crRNA 和四个 crRNA。总而言之，我们的 STU CRISPR 2.0 系统进一步扩展了用于植物基因组编辑和其他应用的 CRISPR 工具箱。
CRISPR-Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species-specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first-generation single transcript unit (STU)-Cas9 system, STU-Cas9-RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU-Cas9 systems and one STU-Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU-Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU-Cas9-Csy4 and STU-Cas9-tRNA systems showed more robust genome editing efficiencies than our first-generation STU-Cas9-RZ system and the conventional mixed dual promoter system. We further applied the STU-Cas9-tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU-based PmCDA1 base editor system is highly efficient in rice. The STU-Cas12a system, based on Cas12a' self-processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.