Melting dsDNA donor molecules potentiates precision genome editing in C. elegans [preprint]
UMass Chan Affiliations
Graduate School of Biomedical SciencesProgram in Molecular Medicine
RNA Therapeutics Institute
Document Type
PreprintPublication Date
2020-08-03Keywords
CRISPRHDR
Genome Editing
Donor DNA
Genetics and Genomics
Investigative Techniques
Molecular Biology
Metadata
Show full item recordAbstract
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.Source
bioRxiv 2020.08.03.235036; doi: https://doi.org/10.1101/2020.08.03.235036. Link to preprint on bioRxiv service.
DOI
10.1101/2020.08.03.235036Permanent Link to this Item
http://hdl.handle.net/20.500.14038/29515Related Resources
This preprint has been published in Genetics, doi:10.1534/genetics.120.303564 and the published version is also available in eScholarship@UMMS.
Rights
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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.Distribution License
http://creativecommons.org/licenses/by-nc/4.0/ae974a485f413a2113503eed53cd6c53
10.1101/2020.08.03.235036
Scopus Count
Except where otherwise noted, this item's license is described as The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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.