University of Massachusetts Medical School Faculty Publications

UMMS Affiliation

RNA Therapeutics Institute; Program in Molecular Medicine; Graduate School of Biomedical Sciences

Publication Date

2020-08-03

Document Type

Article Preprint

Disciplines

Genetics and Genomics | Investigative Techniques | Molecular Biology

Abstract

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.

Keywords

CRISPR, HDR, Genome Editing, Donor DNA

Rights and Permissions

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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

10.1101/2020.08.03.235036

Source

bioRxiv 2020.08.03.235036; doi: https://doi.org/10.1101/2020.08.03.235036. Link to preprint on bioRxiv service.

Journal/Book/Conference Title

bioRxiv

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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