Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Aug 13, 2013Genetics

Accurate and inheritable gene editing in different nematode species using TALENs and CRISPR/Cas9 to create DNA insertions and deletions

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Abstract

Highly effective genome-editing strategies enable precise modifications at targeted DNA locations in nematodes, including species diverged by 300 million years.

Custom-designed nucleases can induce DNA double-strand breaks, triggering either error-prone or precise repair pathways.
Previous techniques only allowed for heritable disruption via random mutagenesis in the Caenorhabditis elegans genome.
New strategies utilize TALE and CRISPR/Cas9 nucleases to create heritable, precise insertions, deletions, or substitutions at specific loci.
These genome-editing tools are applicable to various nematode species, including necromenic and hermaphroditic types.
An efficient pipeline involving germline injection of nucleases and DNA templates allows for targeted genetic modifications and functional tagging.

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Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.

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Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

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