Summary
A bioRxiv preprint uses electronic genome mapping to measure CRISPR-Cas9 target and off-target binding across the human genome in vitro. The study also examines how guide-RNA mismatches and binding-site spacing affect the system.
A bioRxiv preprint reports an electronic genome-mapping method for measuring how CRISPR-Cas9 binds intended and unintended DNA sites across the human genome. The work, posted on September 17, 2026, examines guide-RNA mismatches in vitro and describes a possible use for studying repeat regions associated with genetic disease.
The study was conducted by researchers affiliated with Nabsys. It presents electronic genome mapping (EGM) as a way to observe binding behaviour at single DNA molecules rather than relying only on short synthetic DNA sequences or predicted binding sites.
How electronic genome mapping was used
CRISPR-Cas9 uses a guide RNA, or gRNA, to direct the Cas9 protein to a matching DNA sequence. Target recognition also depends on a nearby protospacer-adjacent motif, commonly called a PAM. A mismatch between the guide RNA and DNA can reduce binding at the intended site, but some mismatches may still permit binding elsewhere in the genome.
EGM detects single-molecule DNA nicks. In the reported experiments, this signal was used to determine where CRISPR-associated complexes bound across the human genome in vitro. The researchers compared perfectly matched guide-RNA sites with sites containing mismatches at different positions along the guide sequence.
The work also used dCas9, a catalytically inactive form of Cas9 that can bind DNA without cutting it, to examine how a bound CRISPR complex interferes with nearby DNA-modifying enzymes. The authors report that bound CRISPR/dCas9 inhibited nickases whose recognition sites were 4–7 base pairs from the gRNA/PAM region.
Binding patterns identified by the study
A guide RNA designed for a sequence next to the FXN repeat expansion associated with Friedreich ataxia bound a single site in the tested genome. By contrast, guide RNAs matching a repetitive sequence bound hundreds of sites. This illustrates how a guide aimed at repeated DNA can produce a much broader binding pattern than one aimed at a unique sequence.
The position of a mismatch also mattered. The PAM and nearby seed-region bases were identified as critical for binding. Single mismatches closer to the guide RNA's 5′ end were more compatible with off-target binding, according to the preprint. These measurements provide a positional view of guide-RNA specificity rather than treating every mismatch as having the same effect.
The authors describe this as the first reported use of EGM to characterise proteins and RNAs, and the first combined application of CRISPR and EGM. They propose that examining many mismatched sites at once could help researchers compare guide designs and optimise Cas proteins or guide RNAs.
The potential disease-related application is primarily experimental detection and characterisation of specific repeat regions. Repeat expansions are difficult genomic regions to analyse, and the study suggests that CRISPR-directed binding combined with EGM could help identify such regions in the human genome.
The evidence is an in-vitro molecular study, not a study in patients. Its findings concern DNA binding and the physical relationship between CRISPR complexes and nearby nickase sites. Whether the same binding patterns translate to cellular DNA or future clinical applications requires further investigation. The preprint also discloses that all authors are current or former Nabsys employees and receive compensation from the company.