An Improved Protocol for High-Efficiency and Cost-Effective CRISPR/Cas9-Mediated Knock-ins in C. elegans
- 1National Centre for Biological Sciences
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.
Related
- Protocol for automated analysis of oil red O-stained Caenorhabditis elegans using Redwormpix, a Python-based application
- Epitope-based labeling for improved live-imaging of endogenous proteins in C. elegans
- Exosome based nanocarriers enabling effective treatment of antimicrobial resistant infections through improved delivery and clinical potential
- Protocol for eVLP-mediated CRISPR-Cas9 and prime editing in pancreatic cancer cell lines
- Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats.
BibTeX
@misc{vats2026improved,
title = {{An Improved Protocol for High-Efficiency and Cost-Effective CRISPR/Cas9-Mediated Knock-ins in C. elegans}},
author = {A. Vats and A. Sen and A. Bandyopadhyay and M. Sudhanand and M. X. Mascarenhas and A. Bhattacharya},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.04.749330},
url = {https://www.biorxiv.org/content/10.64898/2026.09.04.749330v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - An Improved Protocol for High-Efficiency and Cost-Effective CRISPR/Cas9-Mediated Knock-ins in C. elegans
AU - A. Vats
AU - A. Sen
AU - A. Bandyopadhyay
AU - M. Sudhanand
AU - M. X. Mascarenhas
AU - A. Bhattacharya
PY - 2026
DA - 2026/09/04
PB - bioRxiv
DO - 10.64898/2026.09.04.749330
UR - https://www.biorxiv.org/content/10.64898/2026.09.04.749330v1
AB - 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.
ER -