Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

Abstract

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by highthroughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of HermanskyPudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.gene targeting | mouse genetics | knockout | knockin G ene targeting in embryonic stem (ES) cells is routinely applied to modify the mouse genome and establish the mouse as the most commonly used genetic disease model (1). Nevertheless, the production of targeted mutants is a laborious and time-consuming task that requires the construction of targeting vectors with selection markers, the isolation of mutant ES cells, and the generation of germ-line chimaeras. Moreover, advanced allele design strategies, like the creation of missense mutations, require an additional working step to eliminate selection marker genes from targeted loci (2). Therefore, the establishment of such mutants is presently a low-throughput and long-term procedure, restricting rapid advancements in disease modeling. In contrast, a burst of nucleotide replacements, small deletions and insertions in coding regions that underlie Mendelian or complex human diseases are discovered by high-throughput genomic analysis such as whole-exome sequencing (3-5). The faithful reproduction of such mutations in mouse models will be instrumental in understanding disease mechanisms and to develop therapeutic interventions. This demand is neither covered by available mutagenesis technologies nor by large-scale mouse mutagenesis programs that aim for complete gene inactivation in classical or conditional knockout mice (6). To cope with this challenge, we aimed to create mutations within the shortest time directly in the genome of one-cell mouse embryos, avoiding the time-consuming handling of ES cells, gene-targeting vectors, selection markers, and chimaeras. Our approach is based on transcription activator-like (TAL) effector nucleases (TALENs) to create targeted double-strand breaks that are either sealed by homologous recombination (HR) with mutant, synthetic oligodeoxynucleotides (ODNs) or closed by nonhomologous endj...

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

“…We observed TALEN-mediated induction of NHEJ in 25.4% of the offspring independent of targeting construct integration. These results are in line with efficiencies reported for zinc-finger nucleases in mice [25][26][27] and rats 28 , as well as for TALEN-injections in murine oocytes 6,12,13,29 . Newly established methodology using the CRISPR/Cas9 system to induce DNA double-strand breaks and facilitate homologous recombination 21,22 certainly raises high expectations that this system could also be used to mediate homologous recombination of larger donor plasmids directly in oocytes 22 .…”

“…11). While this manuscript was in preparation such findings have been extended to the mouse using a similar strategy for homologous recombination in mice that utilizes either short ssDNA donor templates or donor vectors to correct a single base mutation 12,13 . This work demonstrated that a donor vector with long homology regions, similar to the one used for this study, can be used for homology-directed repair in oocytes to exchange single base mutations.…”

“…Gene targeting via non-homologous end-joining (NHEJ) has also been suggested to generate combined reporter and knockout alleles 9 . Combination of TALEN-based gene targeting with insertion of a few base pairs or transgenes using small ssDNA oligonucleotides or donor vectors with longer homologous arms was initially reported for zebrafish 10,11 and later extended to other animal models including mouse 12,13 . The mouse remains the most widely used animal model in the study of human biology and diseases.…”

Efficient nonmeiotic allele introgression in livestock using custom endonucleases

“…However, indels introduced by NHEJ are variable in size and sequence, which make screening for functionally disrupted clones arduous (15) and do not allow for precise alterations. TALEN-or CRISPR/ cas9-mediated homology-directed repair (HDR) supports the introduction of defined nucleotide changes; however, so far this has only been demonstrated in lower eukaryotic models, including yeast (19), zebrafish (20), and, very recently, mice (21,22).…”

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

“…For the expression of TALENs in mammalian cells we used the expression vector pCAG-TALEN-pA as described in Wefers et al (2013). pT7-TALEN-95A was derived from pCAG-TALEN-pA by replacementof the poly(A) signal sequence with a segment of 95 adenine residues derived from a mouse Oct4 cDNA clone. …”