CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects

Abstract

Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator-Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost-effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off-target effects. Efforts are being made to reduce the off-target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off-target effects. Here, the current GE tools, the off-target effects generated by GE technology, types of off-target effects, mechanisms of off-target effects, major concerns, and outcomes of off-target effects in plants and animals are summarized. The methods to detect off-target effects, tools for single-guide RNA (sgRNA) design, evaluation and prediction of off-target effects, and strategies to increase the on-target efficiency and mitigate the off-target impact on intended genome-editing outcomes are summarized.

Keywords: CRISPR/Cas systems; PAMs; base editing; off‐target effects; sgRNA.