Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes |
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Authors: | Adam D. Norris Hyun-Min Kim Mónica P. Colaiácovo John A. Calarco |
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Affiliation: | *FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138;†Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 |
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Abstract: | Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4–5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments. |
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Keywords: | C. elegans CRISPR Cas9 genome editing |
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