Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

Abstract

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Deploying Genome Editing Tools for Dissecting the Biology of Nut Trees

“…In addition, despite our gRNA selection pipeline being designed to minimize the chance of off-target editing, we cannot rule out this possibility with the current data. This is currently a concern with CRISPR-related technologies and advancements to minimize off-targets are being pursued (Doench et al, 2016;Kleinstiver et al, 2016;Hajiahmadi et al, 2019). In this work, we made a holistic effort to combine filters to minimize the off-target effects for the gRNA selection, a conservative approach that limits the options for gRNA selection but reduces even further the chance of off-targets.…”

A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement

“…However, the off-target issue is a big challenge in the application of gene-editing technology and a recent whole-genome sequencing analysis of CRISPR/Cas9-edited cotton plants revealed rare off-target mutations [198]. The detailed strategy to increase on-target and reduce off-target effects of CRISPR/Cas9 was recently well reviewed [199]. Targeted genome editing in rice using chemically modified donor DNA, which are designed for UTR or prompter region and the homology-directed repair method, was successful [200] and further improvements are expected in the future.…”

In silico Method in CRISPR/Cas System: An Expedite and Powerful Booster

“…Off-target activity leading to mis-editing on the unintended regions had been widely reported, which can trigger unpredictably adverse outcomes ( 104 , 105 ). Undoubtedly, experimental methods including whole-genome sequencing [e.g., CIRCLE-seq ( 106 ), GUIDE-seq ( 107 ), DISCOVER-seq ( 108 ), Digenome-seq ( 109 ), BLESS ( 110 ), and HTGTS ( 111 )] and the improved VIVO strategy ( 112 ) are relatively robust and accurate for off-target identification.…”