From engineering to editing the rat genome

Abstract

Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where ES culture and gene editing technologies can play complementary roles in generating accurate bespoke rat models for studying biological processes and modelling human disease.

Fig. 1

Summary of site-specific nuclease gene editing tools. Site-specific nucleases consist of a DNA-specific binding domain fused to a nuclease domain. In the case of ZFNs and TALENs, the nuclease domain is derived from the FokI restriction endonuclease. The formation of FokI homodimers is required for cleavage to occur. This is achieved by using pairs of ZFNs and TALENs designed to opposite strands and flanking the cut site. CRISPR-mediated cleavage is achieved using the RNA-guided DNA nuclease, Cas9. Following the generation of a DSB, the DNA can be repaired either by NHEJ, which can generate knock-out mutations resulting from the introduction of random insertions or deletions (indels), or more precisely by homology-directed repair using a homologous DNA template containing the desired modification to be inserted

Fig. 2

CRISPR/Cas9-mediated large-scale genomic deletions and replacements. Large-scale genomic deletions or replacements can be achieved using pairs of Cas9/gRNAs designed to flank and excise the region to be deleted/replaced. a For deletions, a single ssODN containing sequence homology to either side of the cleavage site acts as a ‘bridge’ to paste together the non-adjacent ends. b For replacement, a donor plasmid containing the replacement sequence is linearised using a third Cas9/gRNA. Replacement at the desired locus is facilitated by the use of two ssODNs each containing sequence homology to either side of the two cleavage sites bridging the non-adjacent ends