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.
Keywords: CRISPR; CRISPRi/a; Cas9; RNA targeting; gene knock-out; genome editing; guide design; knock-down; knock-in; nickase.
© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.