CRISPR/Cas9 Optimization Using Electronic Genome Mapping: Potential Role for Studying Human Genetic Disease
- 1Nabsys
Abstract
CRISPR Cas9's ability to bind targeted and non-targeted sites and its functional interactions with other proteins and domains have been studied using many technologies. These studies are often limited by using oligos as substrates or by a reliance on predicted binding sites. We show how electronic genome mapping (EGM) with its ability to detect single-molecule DNA nicks can determine how gRNA mismatches impact targeted versus off-target binding across the whole human genome in vitro. Cas9 was found to be capable of binding a single site adjacent to the FXN repeat expansion responsible for Friedreich Ataxia using a gRNA specific for that sequence while gRNAs matching a repetitive sequence were able to bind hundreds of sites. Binding to perfectly matched gRNA sites was compared to sites with mismatches at each gRNA position. The PAM site and nearby seed region bases were confirmed as critical for binding. In contrast, single mismatches toward the gRNA 5' end allowed off-target binding. We also used EGM to determine how physically close the bound dCas9/gRNA complex can be relative to nearby DNA modification sites and still allow activity. Bound CRISPR/dCas9 inhibits nickases with recognition sites 4-7 bp away from the gRNA/PAM. Because EGM can simultaneously evaluate many mismatched off-target sites, these insights may be extended to other CRISPR systems and enable optimization of Cas proteins and gRNAs. This is the first example of EGM being used to characterize proteins and RNAs. This is also the first study demonstrating the combined application of CRISPR and EGM methodology and its potential role in the detection of specific repeat regions in the human genome that are associated with human repeat expansion disorders.
Related
- Tunable Magnetorheological Elastomer Substrates for Studying Mechano-Genetic Interactions in Hypertrophic Cardiomyopathy
- Development of a conceptual apparatus in the field of establishment, formation, conservation, development, studying, and utilization of plant genetic resources collections
- Genetic mapping of Citrullus amarus for resistance to gummy stem blight in watermelon
- Genome-wide mapping of genetic suppression events in Streptococcus pneumoniae
- Mapping human genetic associations to drug targets in valvular heart disease: an integrative evidence synthesis
BibTeX
@misc{opsahl2026crisprcas9,
title = {{CRISPR/Cas9 Optimization Using Electronic Genome Mapping: Potential Role for Studying Human Genetic Disease}},
author = {L. S. Opsahl and W. Huang and S. Koltz and K. L. Mathews and J. F. Thompson},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.14.751601},
url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751601v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - CRISPR/Cas9 Optimization Using Electronic Genome Mapping: Potential Role for Studying Human Genetic Disease
AU - L. S. Opsahl
AU - W. Huang
AU - S. Koltz
AU - K. L. Mathews
AU - J. F. Thompson
PY - 2026
DA - 2026/09/14
PB - bioRxiv
DO - 10.64898/2026.09.14.751601
UR - https://www.biorxiv.org/content/10.64898/2026.09.14.751601v1
AB - CRISPR Cas9's ability to bind targeted and non-targeted sites and its functional interactions with other proteins and domains have been studied using many technologies. These studies are often limited by using oligos as substrates or by a reliance on predicted binding sites. We show how electronic genome mapping (EGM) with its ability to detect single-molecule DNA nicks can determine how gRNA mismatches impact targeted versus off-target binding across the whole human genome in vitro. Cas9 was found to be capable of binding a single site adjacent to the FXN repeat expansion responsible for Friedreich Ataxia using a gRNA specific for that sequence while gRNAs matching a repetitive sequence were able to bind hundreds of sites. Binding to perfectly matched gRNA sites was compared to sites with mismatches at each gRNA position. The PAM site and nearby seed region bases were confirmed as critical for binding. In contrast, single mismatches toward the gRNA 5' end allowed off-target binding. We also used EGM to determine how physically close the bound dCas9/gRNA complex can be relative to nearby DNA modification sites and still allow activity. Bound CRISPR/dCas9 inhibits nickases with recognition sites 4-7 bp away from the gRNA/PAM. Because EGM can simultaneously evaluate many mismatched off-target sites, these insights may be extended to other CRISPR systems and enable optimization of Cas proteins and gRNAs. This is the first example of EGM being used to characterize proteins and RNAs. This is also the first study demonstrating the combined application of CRISPR and EGM methodology and its potential role in the detection of specific repeat regions in the human genome that are associated with human repeat expansion disorders.
ER -