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Page 4 of 13 Li et al. Ageing Neur Dis 2022;2:13 https://dx.doi.org/10.20517/and.2022.13

Figure 2. Flow chart of transgenic and gene editing using SCNT to construct neurodegenerative disease pig models. SCNT: Somatic cell
nuclear transfer.

established [21-24] . This situation was greatly improved with the development of new precise gene editing tools,
which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and
clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas).
ZFNs, composed of DNA-binding domains consisting of tandem zinc finger motifs with nuclease domains
from the endonuclease FokI, can induce the targeted DNA double-stranded breaks (DSBs) that lead to DNA
damage repair mechanisms [25,26] . Although ZFNs have been widely applied in many species, including plants,
animals, and mammalian cells in culture , they have not been used to create large animal models.
[25]

TALENs are an alternative tool for genome engineering [27-29] . They are also fusion proteins of tandem
repeats of a TAL effector protein and the FokI nuclease. TALENs induce the targeted DSBs that activate
[30]
DNA damage response pathways and lead to gene knockout (KO) or knock-in (KI) . As compared with
ZFNs, TALENs are easier to design and synthesize, and some animal models of disease have been
[31]
successfully established using TALENs .
Although ZFNs and TALENs have been applied to various species, CRISPR/Cas9 is now the most widely
used genome editing tool for generating genetically modified animal models. The CRISPR/Cas9 system
confers targeted gene editing by small RNAs that guide the Cas9 nuclease to the target site through base
pairing . When the complex is located at the targeting site of the genome, Cas9 cuts both strands at a
[32]
precise location. Then, the repair mechanism kicks in to rejoin the damaged genomic DNA by non-
homologous end joining (NHEJ) or homology-directed repair (HDR), which may result in mutations to
inactivate or alter gene function. Based on this damage-repair mechanism, scientists have optimized the
CRISPR/Cas9 system to create many genome editing models for small animals, such as mice , rats , and
[34]
[33]
zebrafishes . Large animal models such as pigs have also benefited from this technology. Here, we focus on
[35]
genetically modified pig models of neurological diseases.
Pig models of amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease caused by the
selective death of motor neurons (MNs). With the occurrence of aging, patients with ALS develop