An improved protocol for high-efficiency and cost-effective CRISPR/Cas9-mediated knock-ins in C. elegans

Abstract

CRISPR/Cas9-mediated homology-directed precise genome editing using long single-stranded DNA (ssDNA) donors has expanded the possibilities for generating defined genetic modifications. However, the required ssDNA donor preparation can be technically demanding and often requires extensive locus-specific sequence design. Here, we examined parameters influencing ssDNA donor-mediated genome editing and developed approaches to simplify donor preparation using λ-exonuclease-mediated ssDNA generation. By evaluating donor designs across multiple genomic loci, we found that efficient genome editing can be achieved with relatively short homology regions for a range of insertion sizes. These findings provide a basis for simplifying ssDNA donor preparation and the overall gene-editing pipeline, potentially facilitating its application across species. Key features This protocol offers cost-effective, alternative ssDNA donor preparation strategies, overcoming the requirement of expensive locus-specific, long phosphorylated primers. Efficient genome editing can be achieved with relatively short homology regions for a range of insertion sizes. This protocol provides a simplified CRISPR/Cas9-mediated knock-in pipeline suitable for editing a large number of loci. This protocol is used in: Rosenkranz, N., et al., In situ structure of a gap junction-stomatin complex. Sci Adv, 2025. Vats, A., et al., The combinatorial innexin code of heterochannel electrical synapses governs synaptic function and is maintained by distinct cellular mechanisms. Proc Natl Acad Sci U S A, 2026.