CRISPR Genome Editing Made Easy Through the CHOPCHOP Website

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

VPS13A and VPS13C Influence Lipid Droplet Abundance

“…Human VPS13A and VPS13C knock-outs in U-2 OS cells were generated by two double-stranded breaks (DSBs) using CRISPR/Cas9 approaches. Single-guide RNAs (sgRNA) targeting introns at the 5′ and 3′ sides of exon 2 of VPS13A or VPS13C ( Figure 1 ) were designed using the CHOPCHOP web tool ( Labun et al, 2021 ). The sgRNA oligonucleotides listed in Table 1 were synthesized (IDT), annealed, and ligated into the BbsI site of pSpCas9(BB)-2A-Puro vector (PX459, Addgene #62988).…”

VPS13A and VPS13C influence lipid droplet abundance

“…Human VPS13A and VPS13C knock-outs in U-2 OS cell lines were generated by two double-stranded breaks (DSBs) using CRISPR/Cas9 approaches. Single-guide RNAs (sgRNA) targeting introns at the 5′ and 3′ sides of exon 2 of VPS13A or VPS13C (Figure 1) were designed using the CHOPCHOP web tool (Labun et al, 2021). The sgRNA oligonucleotides listed in Table 1 were synthesized (IDT), annealed, and ligated into the BbsI site of pSpCas9(BB)-2A-Puro vector (PX459, Addgene #62988).…”

RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives

“…Improvements in gRNA design strategies can allow the selection of gRNAs that exhibit minimal off-target effects, with maximal on-target effects. For example, the development of screening algorithms such as GuideScan, CHOPCHOP, and DeepHF have significantly improved the ability to select appropriate and effective gRNAs (Perez et al, 2017;Wang et al, 2019a;Labun et al, 2021). Some comparisons of shRNA and CRISPR-Cas9 systems outlined thus far are available for review.…”