Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases (original) (raw)
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Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins
Scientific reports, 2015
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRN...
Scientific reports, 2017
The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.
Xenotransplantation, 2016
Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig-to-non-human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer (SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3-galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1-KO pigs have successfully been produced by transfecting somatic cells with zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT. Here, we microinjected a CRIS...
Beyond knockout rats: New insights into finer genome manipulation in rats
Cell Cycle, 2011
T he ability to "knockout" specific genes in mice via embryonic stem (ES) cell-based gene-targeting technology has significantly enriched our understanding of gene function in normal and disease phenotypes. Improvements on this original strategy have been developed to enable the manipulation of genomes in a more sophisticated fashion with unprecedented precision. The rat is the model of choice in many areas of scientific investigation despite the lack of rat genetic toolboxes. Most recent advances of zinc finger nucleases (ZFNs) and rat ES cells are diminishing the gap between rat and mouse with respect to reverse genetic approaches. Importantly, the establishment of rat ES cell-based gene targeting technology, in combination with the unique advantages of using rats, provides new, exciting opportunities to create animal models that mimic human diseases more faithfully. We hereby report our recent results concerning finer genetic modifications in the rat, and propose their potential applications in addressing biological questions.
Mouse genome engineering using designer nucleases
Journal of visualized experiments : JoVE, 2014
Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mR...
Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models
Frontiers in Genetics, 2021
The rat has been extensively used as a small animal model. Many genetically engineered rat models have emerged in the last two decades, and the advent of gene-specific nucleases has accelerated their generation in recent years. This review covers the techniques and advances used to generate genetically engineered rat lines and their application to the development of rat models more broadly, such as conditional knockouts and reporter gene strains. In addition, genome-editing techniques that remain to be explored in the rat are discussed. The review also focuses more particularly on two areas in which extensive work has been done: human genetic diseases and immune system analysis. Models are thoroughly described in these two areas and highlight the competitive advantages of rat models over available corresponding mouse versions. The objective of this review is to provide a comprehensive description of the advantages and potential of rat models for addressing specific scientific questi...
An update on targeted gene repair in mammalian cells: methods and mechanisms
Journal of Biomedical Science, 2011
Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA) has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs), small DNA fragments (SDFs), triplexforming oligonucleotides (TFOs), adeno-associated virus vectors (AAVs) and zinc-finger nucleases (ZFNs). Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research.