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Abstract：
The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock-out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock-in Alternate Protocol 2: Design of gRNAs for knock-in using non-homologous end joining Basic Protocol 4: Design of gRNAs for knock-in using Cas9 nickases.
摘要：
用于CRISPR系统的最佳指导RNA(gRNA)序列的设计受到需要在期望的位置实现高效编辑(中靶编辑)而在非预期位置实现最小编辑(脱靶编辑)的挑战。尽管理想情况下实验室验证应用于检测脱靶活动，由于计算预测的速度和低成本，在实践中几乎总是首选。因此，一些研究探索了gRNA-DNA相互作用，以了解CRISPR复合物如何选择其基因组靶标。CHOPCHOP(https://chopchop.cbu.uib.no/）利用这些开发来构建用户友好的Web界面，帮助用户设计最佳的gRNA。CHOPCHOP支持广泛的CRISPR应用，包括基因敲除,序列敲入，和RNA敲除。此外，CHOPCHOP提供可视化，使gRNA的明智选择和支持实验验证。在这些协议中，我们描述了使用CHOPCHOP设计gRNA的最佳实践。©2021作者WileyPeriodicalsLLC出版的当前协议。基本方案1:用于基因敲除的gRNA的设计替代方案1:用于dCas9融合/效应子靶向的gRNA的设计支持方案:用于靶向转基因或质粒序列的gRNA的设计基本方案2:用于RNA靶向的gRNA的设计基本方案3:用于序列敲入的gRNA的设计替代方案2:使用非同源末端连接设计用于敲入的gRNA的设计
