Recent Advances in the Production of Genome-Edited Rats

Abstract

The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.

Keywords: CRISPR/Cas9; GONAD/i-GONAD; TALENs; ZFNs; adeno-associated virus; electroporation; embryonic stem cell; genome editing; microinjection; rats.

Figure 1

Schematic illustration of genome-edited (GE) rat production through microinjection (MI) (A), in vitro electroporation (EP) (B), genome-editing via oviductal nucleic acids delivery (GONAD) (or improved genome-editing via oviductal nucleic acids delivery (i-GONAD)) (C), adeno-associated virus (AAV)-based GONAD, a technique enabling in vitro viral infection of zygotes with recombinant AAV (rAAV) (D), spermatogonial stem cell (SCC)-mediated transgenesis (E), and embryonic stem (ES) cell-mediated transgenesis (F). This figure was drawn in-house, based on the data shown in the paper of Sato et al. [158].

Figure 2

Schematic illustration of single-stranded oligodeoxynucleotide (ssODN)-based deletion of a large fragment spanning a 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. This figure was drawn in-house, based on the data shown in the paper of Yoshimi et al. [46].

Figure 3

Schematic representation of the two-hit by gRNA and two-oligo with plasmid (2H2OP) method for the production of CAG-GFP knock-in (KI) rats generated using the CRISPR/Cas9 system. In the first step, Cas9, together with two gRNAs targeting the rat Rosa26 locus and the CAG promoter in the GFP plasmids, cuts the target sites. In the second step, two ssODNs ligate each cut end to join the genomic DNA and the plasmid DNA via HDR. This figure was drawn in-house, based on the data shown in the paper of Yoshimi et al. [64]. Abbreviations: CAG, chicken β-actin-based promoter; GFP, green fluorescent protein; KI, knock-in; ssODNs, single-stranded oligodeoxynucleotides; pA, poly(A) sites.

Figure 4

Schematic representation of knock-in (KI) experiment in rats toward the mutated Tyr locus performed by Aoshima et al. [141]. The target sequence (exon 2 of Tyr) recognized by crRNA1, 2, and 3 is shown in green. The PAM sequences are underlined. Single-stranded oligodeoxynucleotide (ssODN) (containing wild-type nucleotide “G” that corresponds to mutated nucleotide “A”) is shown in orange below the target sequence. The nucleotide “A/T” marked in red is the mutation causative of the albino phenotype. This figure was drawn in-house, based on the data shown in the paper of Aoshima et al. [141].