Aug 2026· Nature Communications· Vol 17· 0 citations· 115 references
Medicine
TL;DR
It is shown that off-target editing and translocations vary widely between individual cells and organs, motivating the development of more sensitive and organ-specific safety assays for CRISPR therapies.
Abstract
CRISPR-Cas9 holds promise for treating genetic disease, but rare off-target mutations and structural variants remain as key safety concerns, especially at scales relevant to therapy. Here, we establish workflows to resolve Cas9 off-target activity in vitro at single-cell resolution and in vivo across different tissues. Using clonally expanded electroporated mouse embryos and embryonic stem cells, we reveal that individual cells exhibit unique off-target and translocation profiles, including events missed in bulk analyses. Integrating single-cell editing with chromatin accessibility, transcription, and DNA methylation measurements suggest that sequence-independent features modulate Cas9 access and cleavage, with preferential editing in regions characterized by open chromatin and lower methylation. In Cas9-inducible mouse models, editing analyses reveal organ-distinct off-target spectra, DNA repair pathway usage, indel patterns, and markedly varying translocation propensity between tissues. These findings demonstrate that off-target activity is heterogeneous across cells and context-dependent across organs, motivating sensitive single-cell analyses and organ-specific evaluation in preclinical development to more accurately assess risk and improve the safety of CRISPR-based genomic medicines. CRISPR can treat genetic disease, but unintended DNA changes remain a safety concern. Here, authors show that off-target editing and translocations vary widely between individual cells and organs, motivating the development of more sensitive and organ-specific safety assays for CRISPR therapies.
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
+
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 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
A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· bioRxiv· 2 citations
To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· Nature Communications· 0 citations
Abstract A cell’s fate is shaped by its inherited state, or lineage, and the ever-shifting context of its environment. CRISPR-based recording technologies are a promising solution for mapping the lineage of a developing system; however, challenges remain regarding single-cell recovery, engineering complexity, and scale. Here, we introduce BASELINE, which uses base editing to generate high-resolution lineage trees in conjunction with single-cell profiling. BASELINE uses the Cas12a adenine base editor to irreversibly edit nucleotides across target arrays built from 50 synthetic target sites, which are integrated multiple times into a cell’s genome. We demonstrate that BASELINE accumulates lineage-specific marks over a wide range of biologically relevant intervals, recording more than 4300 bits of information in a model of pancreatic cancer, a 50-fold increase over existing technologies. Single-cell sequencing reveals high-fidelity capture of these recorders, averaging 29 cell divisions captured per lineage, within the estimated range of mammalian development. We expect BASELINE to apply to a wide range of lineage-tracing projects in development and disease, especially those in which cellular engineering makes small, more distributed systems challenging.
Evan Winter, F. Emiliani, Aidan M. Cook et al.· Nucleic Acids Research· 0 citations
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
