RNA Therapeutics Institute; Graduate School of Biomedical Sciences
Genetic Phenomena | Genetics and Genomics
CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs-such as green fluorescent protein tags remains challenging in many systems. Here, we describe a streamlined and optimized editing protocol for the nematode Caenorhabditis elegans We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging 14 Argonaute proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based, partially single-stranded, "hybrid" donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci, achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems, and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.
CRISPR, HDR, WormBase, fluorescent tags
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Copyright © 2018 G. A. Dokshin et al. 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
Genetics. 2018 Nov;210(3):781-787. doi: 10.1534/genetics.118.301532. Epub 2018 Sep 13. Link to article on publisher's site
Dokshin, Gregoriy A.; Ghanta, Krishna S.; Piscopo, Katherine M.; and Mello, Craig C., "Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans" (2018). Open Access Articles. 3596.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.