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.
Highlights
The CRISPR system allows for programmable targeting of genomes and transcriptomes, and has been repurposed for a range of applications in biological systems
The guide RNA (gRNA) is a sequence of ∼100-nt that consists of two parts: one that interfaces with the Cas protein and one that guides the complex to its target
When the Cas protein recognizes its preferred protospacer adjacent motif (PAM) in the genome, the adjacent sequence can base-pair with the 20-bp recognition site of the gRNA
Summary
The CRISPR system allows for programmable targeting of genomes and transcriptomes, and has been repurposed for a range of applications in biological systems. Basic Protocol 1 introduces the most basic use of CHOPCHOP to design a gRNA to create a DSB at a specific genomic location for a knock-out. Before using CHOPCHOP, it is important to define your aim (e.g., knock-out of a specific gene) and the required experimental procedure Depending on these choices, you will select the correct Cas protein and the application. Targeting of Cas effectors follows the same principles as Cas targeting for genome editing, but the choice of target is typically different In these experiments, it is often necessary to target a very specific region (e.g., a promoter, enhancer, or splice site), and screening with restriction enzymes and primers is not applicable. The most common application is creating gene knock-outs through the introduction of double-strand breaks followed by repair through the NHEJ pathway This protocol provides a basic outline of the considerations and steps necessary for the use of CHOPCHOP when targeting Cas to genes.
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