靶向基因敲除：用于多重 CRISPR 技术的 gRNA 阵列快速组装的新平台。
PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies.
机构信息
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
出版信息
Cells. 2022 Aug 9;11(16):2467. doi: 10.3390/cells11162467.
Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.
多重 CRISPR 技术在通路工程和基因组编辑方面具有巨大的潜力。然而，它们的应用受到复杂、繁琐和耗时的克隆步骤的限制。在这项研究中，我们开发了一种新方法 PARA，它允许将多达 18 个 gRNA 一步组装到 CRISPR 载体中。在这里，我们证明了 PARA 能够有效地组装 tRNA/Csy4/核酶基 gRNA 阵列。为了辅助这一过程并简化载体构建，我们开发了一个用户友好的 PARAweb 工具，用于设计 PCR 引物和组件 DNA 片段以及模拟组装的 gRNA 阵列和载体序列。
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