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.

CRISPR/Cas9-mediated Angptl8 knockout suppresses plasma triglyceride concentrations and adiposity in rats

“…As noted above, most studies of the function of ANGPTL8 have been performed in mouse models, but if there are indeed interspecies differences, it is important to use other animal models to yield a better understanding of the role of ANGPTL8 in metabolic homeostasis. Rats have long been used in biomedical research, including in the development of drugs, but they have fallen out of favor with the advent of genebased research, as manipulating the rat genome has proven more difficult than working with the mouse genome (20). However, the development of the clustered regulatory interspaced short palindromic repeat (CRISPR) system using CRISPR-associated protein 9 (Cas9) has enabled editing of the rat genome (20,21).…”

“…Rats have long been used in biomedical research, including in the development of drugs, but they have fallen out of favor with the advent of genebased research, as manipulating the rat genome has proven more difficult than working with the mouse genome (20). However, the development of the clustered regulatory interspaced short palindromic repeat (CRISPR) system using CRISPR-associated protein 9 (Cas9) has enabled editing of the rat genome (20,21). In this study, we generated an Angptl8 KO rat using the CRISPR/Cas9 system to investigate the protein's roles in glucose and lipid metabolism.…”

A Stem Cell Reporter for Investigating Pluripotency and Self-Renewal in the Rat

“…Nonetheless, recent advances in understanding the control of self-renewal in ESCs and the development of rationally designed culture systems enabled the first derivation of authentic rat ESCs (Buehr et al, 2008;Li et al, 2008;Ying et al, 2008). Historically, the laboratory rat has been a preferred research animal in many areas of biomedical investigation, including the cardiovascular system and the brain, and this breakthrough in stem cell technology provided a new approach to generate targeted genetic models in this important and useful experimental animal (Meek et al, 2017;Tong et al, 2010). Rat ESCs (rESCs) also provide a unique alternative to mouse ESCs with which to investigate mechanisms regulating pluripotency and self-renewal (Chen et al, 2013;Meek et al, 2013).…”

Rat models of human diseases and related phenotypes: a systematic inventory of the causative genes

“…Efficient methods to generate mutant rats became available; sperm N-ethyl-N-nitrosourea (ENU) mutagenesis followed by gene-targeted screening methods lead to the isolation of several mutants, including knockout (KO) strains [13 and references therein]. Rat ES were successfully derived and could be used for targeted mutations by homologous recombination; more importantly, several methods not relying on the use of ES cells were introduced to generated targeted mutations (often these are KO mutations), namely gene editing by zinc finger nucleases, by transcription activator-like effector nucleases and finally by the clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system [for a review, see 14]. Transgenic rats can also be generated, including humanized rats carrying large chromosomic fragments (“transchromosomic humanized” rats) [15].…”