# Immunodeficient Rabbit Models: History, Current Status and Future Perspectives
## 3 Gene Editing Tools and the Rise of Non-Murine Immunodeficiency Animal Models  
The development of efficient gene editing tools eventually enabled the production of ID animal models in non-murine species. The past decade witnessed the advent of three generations of gene editing nucleases: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and, most recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9). ZFN is the first-generation programmable gene-editing nuclease. In 2009, ZFN was successfully used to achieve efficient gene targeting in mammalian cells [14]. ZFN consists of the zinc finger DNA-binding domains and the DNA cleavage domain of a Type II restriction enzyme Fok1. The zinc finger domains are programmable, and a unique set of zinc fingers can be constructed to target a unique DNA sequence. The DNA cleavage domain Fok1 only works when two Fok1s are paired. Therefore, in a typical ZFN-mediated gene targeting, a pair of ZFNs are designed to flank the target locus such that the two Fok1 domains (one from each ZFN) will meet and induce site-specific double-strand breaks (DSBs) in the DNA, which leads to targeted modification of the genome. TALENs were first reported in 2011 [15; 16]. Like ZFNs, TALENs also work in a pair, where two Fok1s generate DSBs at the target locus in a scissors-like manner. Instead of using zinc fingers, TALENs use TAL DNA binding domains targeting the desired sequence. The TALEN DNA binding domain contains a repeated highly conserved 33-34-amino-acid sequence, except for the 12 th and 13 th amino acids, which are highly variable (repeat variable diesidue, RVD) and show a strong correlation with specific nucleotide recognition [17; 18]. The simple relationship between the amino acid sequence and DNA recognition has made it easy to design and it is relatively practical compared with ZFN technology. Only two years after TALEN was adopted, the third-generation tool, CRISPR/Cas9, emerged and quickly dominated the field [19; 20]. Originally discovered as the bacterial adaptive immune machinery, CRISPR/Cas9 contains two endonuclease domains by itself (i.e., no need to fuse an exogenous nuclease such as Fok1). The most significant difference between CRISPR/Cas9 and its predecessors is that it uses nucleic acid complementation for target recognition. In this way, the tedious protein engineering work to construct sequence-specific zinc fingers or TALs is bypassed. Instead, a complement 20 nucleotide RNA sequence is used, which can be quickly and easily designed and constructed. These programmable nucleases were quickly adapted in animal model development to bypass the need for germline transmitting ESCs [21]. The technical procedure is similar to that of the pronuclear DNA microinjection, which is used for the production of conventionally transgenic animals. Briefly, targeting elements (ZFN, TALEN, or Cas9) are microinjected into pronuclear stage animal embryos in the format of plasmid DNA, mRNA, or protein. The gene-editing nuclease would generate DSBs in the embryo genome; however, the efficiency is very much sequence-dependent. Many of the offspring animals carry mutations at the target locus, which can be used to establish mutant animal lines through routine herd expansion strategies. The production of ID non-murine animal models boomed after 2009. Prior to that, only naturally occurring ID horses and dogs were established through selective breeding [22; 23; 24; 25; 26; 27]. In the past ten years ID rabbits, rats, pigs, hamsters, and non-human primates have been reported from different laboratories across the world (Tables 1 and 2). Among these animal models, ID rats, pigs, and rabbits represent the majority, consistent with the impact of the corresponding model species in the biomedical research community.