Site Specific Mutation of the Zic2 Locus by Microinjection of TALEN mRNA in Mouse CD1, C3H and C57BL/6J Oocytes

Abstract

Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing.

Simultaneous Gene Editing by Injection of mRNAs Encoding Transcription Activator-Like Effector Nucleases into Mouse Zygotes

“…Given the high stringency of site-specific paired TALEN binding, and the requirement for FokI dimerization to initiate DNA cleavage, off-target effects in cultured cells are at best rare and frequently unobserved (14,15,36). Over the last year, several groups have injected TALEN mRNAs directly into fertilized one-cell embryos that, after their transfer to foster mothers, yielded founder mice carrying mutations in any one of several independently targeted genes (23)(24)(25)(26). We adapted this strategy to simultaneously disrupt multiple genes, thereby generating founder mice that carry different combinations of heritable mutated alleles.…”

“…The methods have been used to modify single genes with variable efficiencies (6,(23)(24)(25)(26), but consistent characterization of mutations in founder animals has suffered from ambiguities stemming from different protocols and methods of analysis. We now report the use of a highly efficient TALEN-based strategy for gene editing in mouse zygotes that yields a very high frequency of heritable mutations at the targeted sites.…”

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

“…At these rates one or more targeted alleles can be obtained from a single day of microinjection, typically resulting in 25 pups if FVB-derived embryos are used for microinjection. TALENs have been further used to induce knockout mutations in embryos of the C57BL/6 inbred strains upon cytoplasmic delivery (Davies et al 2013;Sung et al 2013;Qiu et al 2013). Therefore we are confident that it will be also possible to generate targeted mutations by the delivery of TALEN-95A mRNAs and targeting DNA molecules into the pronuclei of C57BL/6 embryos.…”

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the FrogXenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

“…The complex mixture of indels and wild-type alleles we found in many strains likely resulted from a combination of both incomplete cutting and TALEN activity in daughter cells, following division of the single cell progenitor. Similarly, zinc finger nucleases can elicit both heterozygous and homozygous mutations in cultured mammalian cells (Liu et al, 2010;Santiago et al, 2008), and TALEN gene editing in mouse embryos produces heterozygous and mosaic mice (eg, Davies et al, 2013;Sung et al, 2013). To verify clonality, mutant strains were subjected to 2 rounds of clonal dilution and genotyping.…”