CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos

Abstract

Conventional embryonic stem cell (ESC)-based gene targeting, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.

Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis

“…This limitation is because of the technical difficulty in manipulating rat ES cells (26) and germ-line stem cells (27) for targeted genome deletion compared with that in the widely used mouse ES cells (11). In this regard, recent genome-editing technologies, such as ZFN, TALEN, and CRISPR/Cas9, are powerful strategies by which gene knockouts can be generated in various species of animals without using ES cells and homologous recombination (10). Here, we successfully generated genetically modified rats with deletion of Mkx with CRISPR/Cas9.…”

“…In particular, regenerative experiments for tendon repair with precise surgical interventions are challenging, and most reports of cell therapy for tendon repair used animals that were larger than mice (7,9). For physiological experiments, such as treadmill exercise to test the effect of mechanical load on tendons/ligaments, rats are preferable for analyzing the exact responses to the stress because they are physiologically more similar to humans than mice (10). It is also difficult to obtain a sufficient number of primary tenocytes from mice for tendon/ligament research.…”

“…However, technical challenges related to the isolation and culture of ES cells posed difficulties in generating genetically modified rats (10,11). Recent developments in gene-editing technologies, such as zinc-finger nuclease (ZFN) (12), transcription activator-like effector nuclease (TALEN) (13), and clustered regularly interspaced short palindromic repeats/ CRISPR associated proteins (CRISPR/Cas9) facilitate the Significance Molecular mechanisms of tendon development and homeostasis are not well understood.…”

To Crispr And Beyond: The Evolution Of Genome Editing In Stem Cells

“…Fertility and spermatogenesis studies have benefitted from CRISPR-Cas9 SNP interrogation in mouse spermatogonial stem cells [127][128][129]. Additionally, one-cell mouse and rat embryos have been injected with CRISPR-Cas9 components to generate organism-wide genome edits [61,130]. Human stem cells have also been a focus of CRISPR studies, resulting in methodologies facilitating engineering of gene knockouts in human ESC lines and numerous applications in human iPS cells for disease modeling and probing of gene function [47,58,[131][132][133], including the transcriptional programming of neuronal differentiation in iPS cells [134].…”

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene

“…However, optimization of zinc-finger modular arrays is also costly and off-target digestion is also reported (Urnov et al, 2010). Previously, we have shown that the CRISPR/Cas9 system is a powerful and highly efficient technology with less than ten days of hands-on working time (Shao et al, 2014). However, off-targeting excision induced a relatively high frequency of undesired mutations that are a major adverse effect (Pattanayak et al, 2013).…”