Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

ABSTRACT 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.

STRATEGIES to engineer heritable, site-directed mutations at endogenous loci have revolutionized our approach to- ward manipulating and dissecting genome function. Studies of plants and animals alike, whether conducted in whole organ- isms or cell lines, have benefitted gready from these genome- editing approaches (Bibikova et al. 2002; Beumer et al. 2006; Doyon et al. 2008; Geurts et al. 2009; Hockemeyer et al. 2009; Holt et al. 2010; Zhang et al. 2010; Hockemeyer et aL 2011; Tesson et al. 2011; Wood et al. 2011; Young et aL 2011; Bedell et aL 2012; Bassett et al. 2013; Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013; Wang et al. 2013; Zu et al. 2013). The most modem tools for modifying complex genomes at single-nucleotide resolution are site-specific nucleases that induce DNA double-strand breaks (DSBs) at specifically des- ignated genomic locations. DSBs trigger repair pathways that can elicit targeted genetic reprogramming, primarily through two mechanisms: error-prone nonhomologous end joining (NHEJ) (Lieber 2010) and precise, homology-directed recom- bination or repair (HDR) (Chapman et al. 2012). NHEJ re- joins broken ends of chromosomes through an imprecise process that produces nucleotide insertions, deletions, and indels at the DSB site, causing random, heritable null or partial loss-of-function mutations. In contrast, HDR uses homologous DNA as a template to create accurate repairs, guided either by a sister chromosome or by introduced ho- mologous DNA to generate specific deletions or insertions in predictable ways (Chen et al. 2011; Bedell et al. 2012). DSBs made by any site-specific nucleases should possess the capacity to enter the homology-directed repair pathway and generate precisely specified changes in the genome.

Effective nucleases with engineered specificity include the zinc-finger nucleases (ZFNs), the transcription activator- like effector (TALE) nucleases (TALENs), and the RNA- guided CRISPR-associated (Cas9) endonuclease (Urnov et al. 2010; Bogdanove and Voytas 2011; Jinek et al. 2012; Wiedenheft et al. 2012; Gaj et al. 2013; Wei et al. 2013). ZFNs and TALENs contain fusions between the DNA cleavage domain of the FokI endonuclease and a custom- designed DNA-binding domain: either C2H2 zinc-finger motifs for ZFNs or TALE domains for TALENs (Urnov et al. …

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