An improved protocol for high-efficiency and cost-effective CRISPR/Cas9-mediated knock-ins in C. elegans
Listed in
This article is not in any list yet, why not save it to one of your lists.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.
