An Improved Protocol for High-Efficiency and Cost-Effective CRISPR/Cas9-Mediated Knock-ins in C. elegans

Authors

Vats, A., Sen, A., Bandyopadhyay, A., Sudhanand, M., Mascarenhas, M. X., Bhattacharya, A.

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 {lambda}-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.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 08 Sep 2026.

Stats

1-terrible, 9-excellent. How would you rate this preprint? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.