RNA Therapeutics Institute; Program in Molecular Medicine; Graduate School of Biomedical Sciences
Genetics and Genomics | Investigative Techniques | Molecular Biology
CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.
CRISPR, HDR, Genome Editing, Donor DNA
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
DOI of Published Version
bioRxiv 2020.08.03.235036; doi: https://doi.org/10.1101/2020.08.03.235036. Link to preprint on bioRxiv service.
Ghanta KS, Mello CC. (2020). Melting dsDNA donor molecules potentiates precision genome editing in C. elegans [preprint]. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/2020.08.03.235036. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1738
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This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License