Molecular mechanisms, off‐target activities, and clinical potentials of genome editing systems

Abstract

Methodologies of genome editing are rapidly developing with the improvement of gene science and technology, mechanism‐based understanding, and urgent needs. In addition to the specificity and efficiency of on‐target sites, one of the most important issues is to find and avoid off‐targets before clinical application of gene editing as a therapy. Various algorithms, modified nucleases, and delivery vectors are developed to localize and minimize off‐target sites. The present review aimed to clarify off‐targets of various genome editing and explore potentials of clinical application by understanding structures, mechanisms, clinical applications, and off‐target activities of genome editing systems, including CRISPR/Cas9, CRISPR/Cas12a, zinc finger nucleases, transcription activator‐like effector nucleases, meganucleases, and recent developments. Current genome editing in cancer therapy mainly targeted immune systems in tumor microenvironment by ex vivo modification of the immune cells in phases I/II of clinical trials. We believe that genome editing will be the critical part of clinical precision medicine strategy and multidisciplinary therapy strategy by integrating gene sequencing, clinical transomics, and single cell biomedicine. There is an urgent need to develop on/off‐target‐specific biomarkers to monitor the efficacy and side‐effects of gene therapy. Thus, the genome editing will be an alternative of clinical therapies for cancer with the rapid development of methodology and an important part of clinical precision medicine strategy.

A CRISPR/CAS9‐based strategy targets the personalized chimeric neosequence in fusion‐driven cancer genome for precision medicine

“…1 With the development of genome editing approaches, new possibilities for directly targeting the genomic sequence of cancer cells have arisen. Remarkably, CRISPR/CAS9 nuclease-based genome editing is a well-suited tool to target cancercausing mutations, 2,3 including single nucleotide polymorphisms (SNPs) and short insertions/deletions (indels), 4 as they can create new protospacer adjacent motif (PAM) sequences. The genomic breakpoints of fusion genes are more suitable for CRISPR/CAS9 targeting than SNPs and short indels due to the wide range of PAM appearance and the high tolerance of base mismatch ( Figure 1A).…”

Spatiotemporal molecular medicine: A new era of clinical and translational medicine

“…With the improvement of on-/off-target site specificity and efficiency and improved understanding of structures, mechanisms, clinical applications, and offtarget activities of genome editing systems, genome editing will become one of the clinical precision medicine strategies and multidisciplinary therapy strategies by integrating gene sequencing, clinical trans-omics, and single-cell biomedicine. 15 As parts of spatiotemporal molecular medicine, spatial transcriptomics, proteomics, metabolomics, and bioinformatics can be the important tools to evaluate whether the body is suitable for targeted gene editing by programmable endonucleases prior to the treatment and monitor how the systems' responses occur and how spatiotemporal controls might regulate functional switches or be regulated by gene editing associated alterations after the gene therapy. 16,17 Spatiotemporal molecular medicine requires the obvious contributions from rapid development of artificial intelligence, automatic robots, and computational and mathematical models.…”

A refocus on the advances of single-cell biomedicine

“…Target-oriented regulations and associated networks demonstrated cell type-specific expressions of target genes and its regulated genes in a clear gene-controlled cell. Post-transcriptional profiles of the single cells are also a critical part in single-cell biomedicine in cell biology and toxicology (Qiao and Wang 2019 ; Gil et al 2019 ; Zheng et al 2020 ). It is important to define the relative abundances of protein products, post-translational modifications, protein localization, turnover, protein interactions, and protein function at a single-cell level to design proteomic profiles of body fluids and tissues that can identify biomarkers and therapeutic targets.…”

“…Gene editing can be an alternative for new therapies with the specificity and efficiency of on-target sites. On the other hand, off-targets of various genome editing should be emphasized for clinical application (Wang et al 2019 ; Wang and Wang 2019 ; Zheng et al 2020 ).…”