Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases

By Y. Doyon, J. McCammon, Jeffrey C. Miller, F. Faraji, C. Ngo, George E. Katibah, Rainier Amora, Toby Dylan Hocking, Lei Zhang, E. Rebar and 3 more. Nature Biotechnology, 2008

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We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast–based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/ceb3b3ed20ec016dc4d7cf63cf1be5053b03b355

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Citations in the corpus

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing? (2019, methodology): “Genome editing using targetable nucleases such as ZFNs (Bibikova et al., 2002; Doyon et al., 2008; Geurts et al., 2009; Urnov et al., 2010), TALENs (Huang et al., 2011; Tesson et al., 2011; Joung and Sander, 2013) and CRISPR/Cas9 (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013a; Hwang et a”
mRNA-based therapeutics — developing a new class of drugs (2014, neutral): “IVT mRNAs encoding ZFNs, TALENs and Cas9 have been applied successfully to edit genomes by disrupting or integrating sequences ex vivo in embryonic cells from different species (for example, mice, rats and rabbits) and in vivo in rodents 168–175 .”
Zebrafish: Development of a Vertebrate Model Organism (2018, background): “…then introducing short indels (typically resulting in a loss-of-function frameshift) or homology-mediated repair allowing a simultaneously injected sequence to be placed at the site of the break (Bedell et al., 2012; Dahlem et al., 2012; Doyon et al., 2008; Foley et al., 2009; Zu et al., 2013).”
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (2011, background): “Accordingly, designer nuclease-based genome engineering has been successfully established in more than 10 model organisms thus far, including plants (3,4), invertebrates (5), fish (6,7) and mammals (8).”
Zinc finger nuclease-mediated CCR5 knockout hematopoietic stem cell transplantation controls HIV-1 in vivo (2010, neutral): “ZFNs can be designed to bind to a specific genomic DNA sequence and effect permanent knockout of the targeted gen”
Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection (2011, methodology): “DNA encoding CCR5-224 or VF2468 ZFNs were cloned into a CMV-driven mammalian expression vector in which both ZFN monomers were translated from the same mRNA transcript in stoichiometric quantities using a self-cleaving T2A peptide sequence similar to a previously described vecto”
The discovery of zinc fingers and their applications in gene regulation and genome manipulation. (2010, background): “(53) have shown that heritable mutations can be produced in zebrafish by generating mRNAs encoding ZFNs for the locus of interest and injecting them into embryos at the one-cell stage.”
Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish (2014, methodology): “Initial work utilized zinc finger nucleases (ZFNs; Doyon et al., 2008; Meng et al., 2008), and transcription activator-like effector nucleases (TALENs), which provide improved specificity over ZFNs, also have been developed (Cade et al.”
Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations (2011, neutral): “ZFNs against the human a-synuclein locus were designed using an archive of prevalidated two-finger modules, as described (Doyon et al., 2008; Miller et al., 2007; Perez et al., 2008; Urnov et al., 2005).”
Distinct factors control histone variant H3.3 localization at specific genomic regions (2010, methodology): “3B gene were designed with an archive of validated two-finger modules (Doyon et al., 2008; Urnov et al., 2005).”