In vivo genome editing targeted towards the female reproductive system.

The discovery of sequence specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 has revolutionized genome editing. The CRISPR/Cas9 system has particularly emerged as a highly simple and efficient approach towards generating genome-edited animal models of most of the experimental species. The limitation of these novel genome editing tools is that, till date, they depend on traditional pronuclear injection (PI)-based transgenic technologies developed over the last three decades. PI requires expensive micromanipulator systems and the equipment operators must possess a high level of skill. Therefore, since the establishment of PI-based transgenesis, various research groups worldwide have attempted to develop alternative and simple gene delivery methods. However, owing to the failure of chromosomal integration of the transgene, none of these methods gained the level of confidence as that by the PI method in order to be adapted as a routine approach. The recently developed genome editing systems do not require complicated techniques. Therefore, presently, attention is being focused on non-PI-based gene delivery into germ cells for simple and rapid production of genetically engineered animals. For example, a few reports during the previous 1-2 years demonstrated the use of electroporation (EP) in isolated zygotes that helped to overcome the absolute dependency on PI techniques. Recently, another breakthrough technology called genome editing via oviductal nucleic acids delivery (GONAD) that directly delivers nucleic acids into zygotes within the oviducts in situ was developed. This technology completely relieves the bottlenecks of animal transgenesis as it does not require PI and ex vivo handling of embryos. This review discusses in detail the in vivo gene delivery methods targeted towards female reproductive tissues as these methods that have been developed over the past 2-3 decades can now be re-evaluated for their suitability to deliver the CRISPR/Cas9 components to produce transgenic animals. This review also provides an overview of the latest advances in CRISPR-enabled delivery technologies that have caused paradigm shifts in animal transgenesis methodologies.