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Genetics. 2018 Nov;210(3):781-787. doi: 10.1534/genetics.118.301532. Epub 2018 Sep 13.

Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans.

Author information

1
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
2
Howard Hughes Medical Institute, Worcester, Massachusetts 01605.
3
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 craig.mello@umassmed.edu.

Abstract

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.

KEYWORDS:

CRISPR; HDR; WormBase; fluorescent tags

PMID:
30213854
PMCID:
PMC6218216
DOI:
10.1534/genetics.118.301532
[Indexed for MEDLINE]
Free PMC Article

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