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Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans
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Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans
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Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans
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Journal Article
Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans
2020
Overview
Abstract
Melting and fast cooling double stranded DNA donor molecules prior to injection dramatically increases the frequency of homology-directed repair for edits such as insertions of fluorescent protein markers in Caenorhabditis elegans. Strategies described here enable consistently ...
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 coselection 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.
