RNA Therapeutics Institute; Program in Molecular Medicine; Graduate School of Biomedical Sciences
Genomics | Molecular Biology | Molecular Genetics
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, Donor DNA, Genome Editing, HDR
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Copyright © 2020 Ghanta and Mello. Available freely online through the author-supported open access option. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
DOI of Published Version
Ghanta KS, Mello CC. Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Genetics. 2020 Nov;216(3):643-650. doi: 10.1534/genetics.120.303564. Epub 2020 Sep 22. PMID: 32963112; PMCID: PMC7648581. Link to article on publisher's site
Ghanta KS, Mello CC. (2020). Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Open Access Publications by UMMS Authors. https://doi.org/10.1534/genetics.120.303564. Retrieved from https://escholarship.umassmed.edu/oapubs/4358
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.