Jul 2026· Theoretical and Natural Science· Vol 182, pp. 207-216· 0 citations
TL;DR
This review provides a thorough overview of the current state of knowledge on off-target causes, detection and prediction methods, and mitigation strategies for CRISPR-Cas9 applications, and integrates these dimensions to provide a clear framework for controlling when, where and how CRISPR-Cas9-mediated alterations occur.
Abstract
One of the most efficient and powerful genome-editing instruments, CRISPR-Cas9 represents a high promise in treating disease and examining the genome. Nevertheless, its clinical translation is limited by off-target effects that cause harmful genomic mutations and potentially fatal safety issues. This review provides a thorough overview of the current state of knowledge on off-target causes, detection and prediction methods, and mitigation strategies. MAIN REASONSThese include mismatch tolerance of Cas9-gRNA and sustained expression associated with some delivery methods. Finally, the detection and prediction tools based on representative approaches such as GUIDE-seq, CIRCLE-seq, Cas-OFFinder and DeepCRISPR are evaluated and their advantages and limitations are listed. The mitigation strategies such as sgRNA engineering, Cas9 protein engineering and transient delivery systems are also discussed. The study argues that the data mentioned above only measures an important first step, and that the realization of clinically relevant detection models that reliably mimic in vivo conditions is critical to screening safety. This review integrates these dimensions, so as to offer a clear framework for controlling when, where and how. Integrating these dimensions allows this review to provide a clear framework for controlling when, where and how CRISPR-Cas9-mediated alterations occur afford systemic implementation strategies that improve the safety of CRISPR-Cas9 applications.
Accurate detection of off-target activity in primary human cells is crucial for ensuring the safety of gene therapies, yet existing methods often lack sufficient sensitivity. To address this limitation, we develop Tracking-seq2, an advanced technology that integrates exogenous 5′ → 3′ exonuclease treatment and non-homologous end joining (NHEJ) pathway inhibitors with the original Tracking-seq. Tracking-seq2 exhibits enhanced sensitivity in profiling off-target sites of diverse genome editors—including Cas9, Cas12a, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Critically, Tracking-seq2 is directly applicable to clinically relevant primary human cell types, such as T cells and CD34
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hematopoietic stem and progenitor cells (HSPCs). Furthermore, our findings reveal that genomic variations drive distinct off-target heterogeneity across different individuals, highlighting the necessity for personalized safety assessment in clinical genome editing applications. Tracking-seq2 provides a robust platform for sensitive off-target detection in primary cells, with sensitivity comparable to or exceeding current state-of-the-art methods.
Accurate identification of CRISPR-Cas9 off-target sites is essential for the safety assessment of genome-editing-based therapies. While numerous in silico prediction tools have been developed, their comparative performance and practical utility in preclinical workflows remain incompletely defined. We performed a systematic benchmarking of 14 in silico CRISPR-Cas9 off-target prediction tools, including both standard approaches and machine learning-based models. The analysis was based on a curated dataset derived from the CRISPRoffT database, comprising 3,827 deep-sequenced genomic sites across 26 guide RNA/Cas9 combinations in human cells. Sites with indel frequencies ≥0.1% were operationally defined as true off-targets. We evaluated tool performance using score distributions, correlation with indel frequencies, precision-recall characteristics, recall among top-ranked candidate sites, and the effect of combining tools. All tools assigned higher scores to true off-target sites compared with nontarget sites, although substantial overlap between classes was observed. Correlation between prediction scores and indel frequencies was weak to moderate, indicating limited ability to predict editing magnitude. Precision-recall performance was moderate across all tools, reflecting inherent trade-offs between sensitivity and specificity. Recall increased with the number of predicted sites considered, reaching approximately 77% among the top 500 and up to 83% among the top 1,250 sites, but leaving a substantial fraction of true off-targets undetected. Combining tools yielded only modest improvements. Current in silico tools enable prioritization of CRISPR-Cas9 off-target candidates but remain limited in their ability to comprehensively identify and quantitatively predict off-target activity. Our findings highlight the importance of considering both ranking performance and candidate site coverage and support the use of combined computational and experimental strategies for robust off-target assessment in preclinical gene editing workflows.
M. M. Kaufmann, Maren Hackenberg, William Jobson Pargeter et al.· Human Gene Therapy· 0 citations
A major focus of this review is the inclusion of recent hybrid systems (VLPs, SORT-LNPs) and the recognition that chemical modification of guide RNAs is a critical parameter for therapeutic success and that hybrid systems and stimuli-responsive nanoparticles are poised to dominate the next 5 years of clinical development.
M. Rezaee, F. Izadi, Saeed Nobaharian et al.· Beni-Suef University Journal...· 0 citations
This review focuses on analytical and translational frameworks for CRISPR fidelity assessment, with emphasis on the strengths and limitations of current bioanalytical platforms.
Arpita Mukherjee· Journal of Rare Diseases· 1 citation
CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific single-guide RNAs for the design of compact libraries. While several genome-wide CRISPRi-Cas9 libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets, and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed-sequence patterns. We leverage these findings to design an optimized CRISPRi-Cas9 library, Katsano, and validate its performance with genome-wide viability screens.
Smriti Srikanth, Fengyi Zheng, Laura M Drepanos et al.· Cell Genomics· 0 citations
The current developments in engineering of Cas effector protein, optimizing guide RNA designs, developing advanced reporter probes, and utilizing chemical additives are summarized and their significant contribution to increasing the fidelity of editing and diagnostic sensitivity is highlighted.
Sheng Li, Han Li, Bojie Chen et al.· Interdisciplinary Medicine· 0 citations