This review focuses on analytical and translational frameworks for CRISPR fidelity assessment, with emphasis on the strengths and limitations of current bioanalytical platforms.
Abstract
The clinical translation of CRISPR- Cas9 therapeutics requires rigorous and biologically grounded evaluation of genome editing fidelity that extends beyond conventional measures of on-target efficiency and canonical off-target detection. Existing fidelity assessment strategies often capture limited aspects of editing outcomes and do not fully account for context-dependent biochemical, epigenetic, and cellular heterogeneity that can influence therapeutic safety and efficacy. This review focuses on analytical and translational frameworks for CRISPR fidelity assessment, with emphasis on the strengths and limitations of current bioanalytical platforms. Recent advances integrating orthogonal technologies including digital droplet PCR, genome-wide off-target mapping approaches (e.g., CIRCLE-seq and GUIDE-seq), and single-cell proteogenomic profiling enable more comprehensive characterization of editing outcomes across molecular and functional dimensions. Applications in edited T cells targeting immune checkpoints and in hematopoietic stem cell correction models illustrate how multi-layered analyses can reveal context-dependent variability and previously undetected off-target effects. Emerging approaches, including integrative scoring frameworks and AI-assisted analytical models, are discussed in an exploratory context, with emphasis on current limitations, validation requirements, and challenges in standardization. In addition, ongoing efforts toward minimally invasive monitoring strategies are considered with respect to their potential, as well as their current constraints. Despite significant progress, major challenges remain in assay harmonization, cross-platform comparability, and translation to clinically actionable frameworks. Accordingly, CRISPR fidelity is best understood as a multi-dimensional biological phenotype that requires careful, context-specific evaluation. This perspective provides a balanced foundation for advancing genome editing toward safer and more reliable therapeutic applications.
A comprehensive review of delivery modalities of CRISPR systems solely in vivo that underpin their therapeutic translation and outlines the remaining barriers to durable, tissue-selective, and broadly deployable CRISPR therapeutics is provided.
L. Martin, Jure Bohinc, Alessandra Recchia et al.· International Journal on Bio...· 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
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.
This article synthesizes contemporary advancements in CRISPR-mediated mammalian genome modification, detailing core mechanisms – such as guide RNA and the Cas9 endonuclease – alongside next-generation modalities, including base and prime editing.
Olga Aldoshina, Dmitriy Lazarev, E. Smirnova· Veterinariya, Zootekhniya i...· 0 citations
CRISPR has progressed from an experimental genome-engineering technology to a clinically relevant therapeutic platform, although its future impact will depend on the ability to combine molecular precision and durable therapeutic benefit with rigorous safety assessment, responsible governance, and equitable access across diverse populations and healthcare systems.
G. Alejandro, Ortega Moreno, G. Amaya et al.· International science journa...· 0 citations
CRISPR has emerged as a next-generation gene-editing tool with the potential to target the molecular pathways associated with ageing and related disorders. It functions through RNA-guided Cas nucleases, directing DNA cleavage and utilizing the native DNA repair machinery for genetic manipulations. Advances in CRISPR technology have significantly enhanced the precision and flexibility of techniques for genome editing. The enzyme Cas9's ability to cut DNA at exact site has revolutionized genome editing by enabling accurate modifications within living eukaryotic cells. This review critically examines recent developments in CRISPR-based technologies, including Cas9, Cas12, base editing, prime editing, and CRISPR-mediated gene regulation. It highlights their rising applications in ageing research, with more emphasis on neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The review also discusses the major pharmacological and translational challenges that currently limit clinical applications, including inefficient tissue-specific delivery, off-target genome editing, immunogenicity, manufacturing complexity, and long-term safety concerns. Also, recent progress in both, viral and non-viral delivery methods are critically evaluated, including adeno-associated viruses, lentivirus vectors, lipid nanoparticles, gold nanoparticles, exosomes, electroporation, and microinjection, is thoroughly discussed to highlight their therapeutic potential and translational limitations. Current studies indicate that CRISPR-based approaches have preclinical potential for targeting important hallmarks of ageing, particularly genomic instability, telomere attrition, and mitochondrial dysfunction. Other hallmarks of ageing, such as stem cell exhaustion, epigenetic modifications, and microbiome changes, are at earlier stages of development. Overall, this review describes future strategies for developing safe, precise, and clinically translatable CRISPR-based treatments to promote healthy ageing.
Sakshi Rathore, Akash Gupta, Kamal Shah et al.· Ageing Research Reviews· 0 citations