Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

Abstract

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1 ) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk

“…This replacement may hinder the exchange between the endogenous sequences and the donor template because ssODNs can function as effective templates for HR but are generally 40–200 nt in length. A previous study that used a DNA vector as a template showed that the distance does not affect the targeting efficiency in rat zygotes34. Consistent with this finding, the present study showed that the efficiency of HR using homologous arms contiguous to the DSB site (~17 bp) was similar to the efficiency when the homology was ~80 bp from the DSB site in vitro .…”

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

“…S3b,c). Previous studies have suggested that linearized templates are more efficient for HDR integration when compared to circular templates3031. Therefore, we created a “self-linearizing” minicircle exchange template, as it possessed the same gRNA sites (G10 and G13) present in the MHC locus, and thus this template would be linearized in situ (after transfection) by Cas9.…”

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats

“…and mice (Kisseberth et al, 1999;Remy et al, 2014;Kobayashi et al, 2012;Tsuchida et al, 2016). Like in LSL-Cas9 rats, the LSL-nickase gene targeting construct was designed to mediate transgene expression upon removal of the floxed transcription terminator signals by Cre recombinase ( Figure 7A).…”