## 3 History of the Methods to Produce Transgenic Rabbits
### Novel Genome Editing Technologies to Generate Knock-In (Transgenic) Rabbits  
In the past decade, the emergence of novel genome editing technologies, such as ZNF, TALEN, and CRISPR/Cas9, has dramatically changed the rate of progress in current biomedical science. In 2012, [PERSON] et al. [43] found that Cas9, which is a component of the CRISPR/Cas system, is a DNA endonuclease dependent on RNA, which generates site-specific double-stranded DNA breaks (DSBs). In 2013, several groups succeeded in genome engineering of mammalian cultured cells using the CRISPR/Cas9 system [44; 45]. This technique allows deletion and displacement of a specific DNA sequence with high efficiency and ease. The genome editing by the CRISPR/Cas9 system is much simpler and easier than ZNF and TALEN, and shows high efficiency. It enables the generation of knockout animals using fertilized eggs of any species without established ES cells.  
CRISPR was discovered as an acquired immunity system (i.e., an exclusion system for foreign DNA) in _Eubacterium_ and _Archaebacterium_. The Cas9 protein forms a complex with gRNA that has a homology sequence of target DNA and induces a site-specific DSB. In the CRISPR/Cas9 system, it is only 20 bases in the gRNA of approximately 100 bases, which prescribe the target sequence of the DSB. Therefore, only 20 bases need to be changed for each target and all other base sequences and Cas9 are common. Additionally, the gRNA can be easily obtained from a manufacturing company. As a result of these reasons, the CRISPR/Cas9 system underwent rapid widespread use, even by inexperienced researchers, in molecular biology experiments as a genome editing tool.  
The genome editing by CRISPR/Cas9 has potential benefits to not only generate knockout animals, but also produce knock-in animals by gene integration into specific DSB sites of host DNA when the donor ssDNA (subject genetic sequence including the homology arm of both sides) is added to gRNA and Cas9 mRNA or Cas9 protein at microinjection into embryos. The integration of donor DNA was limited to the small single-stranded oligodeoxynucleotide (ssODN) and the insertion efficiency was low at first, but the integration rate of kb-based ssDNA has increased owing to the improvement of genome editing technology. Recently, [PERSON] et al. [16] reported that RS-1, a homologous recombination (HDR) enhancer, increases the efficiency of producing knock-in rabbits through the genome editing technique. They knocked in enhanced green fluorescent protein (EGFP) and tdTomato genes into the Rosa26 locus of the rabbit by this method and reported knock-in efficiencies of 26.3-35.0% of total born rabbits and 6.8-7.0% of the total injected embryos [16; 29]. Using this method, the knock-in efficiency should be more than 10-fold that of traditional pronuclear microinjection.  
In 2014, [PERSON] et al. [46] reported successful introduction of endonuclease mRNAs into zygotes to induce editing at targeted loci and efficiently produced knockout rats by the electroporation method with a high survival rate. It is noteworthy that electroporation of ZFNs resulted in an embryonic survival rate of 91% and a genome editing rate of 73%. This method has some advantages compared with the traditional microinjection technique. Using this method, specialized skills for microinjection are not needed and a large number of zygotes can be manipulated at one time. The electroporation technique may accelerate the production of genetically modified animals because of its simplicity and ease. New technologies and techniques applicable to the production of genetically modified animals are developing and improving daily.