Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Abstract

Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine.

Keywords: CRISPR/Cas9; base editors; gene targeting; homology dependent repair; non-homologous end joining repair pathway; targeting specificity.

Figure 1

Effects of off-target mutation on animal and plant phenotype. Off-target causes genetic mutations. In clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system gRNA sometimes binds other than target loci, off-target site. It may activate the oncogenes that initiate tumor cell formation in the animal body or may change gene function that leads to undesirable phenotypic expression (sensitive to diseases) in plants. The figure has been created with BioRender.com.

Figure 2

Deactivated Cas9 (dCas9) used for gene activation, repression and base editing. (A) Activations domain such as VP64, P65AD, VPR, Rta fuse to dCas9 to re-activate the gene expression; (B) repression domains such as KRAB, DNMT, LSD1 and HDAC fuse to dCas9 to repress/decrease the gene expression; (C) APOBEC1 (cytidine deaminase) and ABE (adenosine deaminase) fuse to dCas9 for efficient base editing. The figure has been created with BioRender.com.