Biology subjects

Selfjord, N.

Publications and source records attributed to Selfjord, N..

2 recordsLinked to original sources

bioRxiv2026

Single-Cell and Tissue-Specific CRISPR Editing Analyses Unveil New Insights to Off-Targets and Translocations

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. We established 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 suggested 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 revealed 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.

genomics↗

bioRxiv2024

Improved nuclease-based prime editing by DNA repair modulation and pegRNA engineering

Prime editing is a genome engineering tool that allows installation of small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing nuclease (PEn) utilizes a fully active Cas9 instead of the nickase employed in conventional prime editors. PEn is capable of editing sites resistant to nickase-based prime editors but induces more undesired editing events. In this work, we introduce two strategies to enhance PEn precision and efficiency. First, we apply a small molecule approach, selectively modulating DNA repair pathways, to improve PEn precision up to 9.8-fold and reduce off-target editing by 90%. Second, through pegRNA engineering, we devise a strategy that mitigates unintended pegRNA scaffold integration, which is a common prime editing by-product, enhancing precision up to 3.5-fold. We apply this approach to a specific type of PEn editing mediated through non-homologous end joining and use it to achieve efficient and precise prime editing in multiple human cell lines, primary human hepatocytes, and mouse embryos. Together, this work presents two general strategies to improve prime editing, overcomes the limitations of current PEn editors, and provides reliable and precise genome editing outcomes, a pivotal requirement for therapeutic applications.

bioengineering↗