Dual-single-guide RNA strategy improves CRISPR-mediated homology-directed repair in Aspergillus

TL;DR

This paper introduces a new CRISPR strategy to improve gene editing efficiency in fungi by aligning donor DNA with the target site.

Contribution

A dual-single-guide RNA design that enhances homology-directed repair by creating a geometry-matched strand-invasion window.

Findings

Integration efficiency drops when insertion sites are misaligned with the strand-invasion path.

The dual-single-guide RNA design significantly improves knock-in outcomes across various editing tasks.

The strategy generalizes across multiple fungal species and supports precise genome engineering.

Abstract

CRISPR–Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3′ ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks—including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis—and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.

Graphical Abstract

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