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Li et al. Ageing Neur Dis 2022;2:13 https://dx.doi.org/10.20517/and.2022.13 Page 7 of 13

[59]
18 months, which may represent an early event in the pathogenesis of AD . In 2017, another group used a
retroviral multi-cistronic vector to generate an AD transgenic pig carrying three AD-related genes with a
total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and
hPS1 (M146V and L286P). They confirmed that transgenes were expressed at especially high levels in the
brain. The levels of Aβ-40/42, total Tau, and GFAP were high in the brains of these transgenic animals as
well. They proposed that more tests are needed in the future to find out if these pigs have age-dependent
phenotypes of AD .
[60]

Pig models of Parkinson’s disease
Parkinson’s disease (PD), characterized by slowness of movement, limb stiffness, and tremors, is the second
most common neurodegenerative disorder in the world. PD patients may also have issues such as cognitive
issues, depression, anxiety, olfactory loss, and gastrointestinal disorder. The motor symptoms of PD are
[61]
caused by the death of dopaminergic neurons in the substantia nigra . Loss of dopamine neurons causes a
[62]
drop in dopamine levels in the striatum, which leads to disrupted motor control . Many mutations or
variants in a number of genes, such as α-synuclein (SNCA), leucine-rich repeat kinase 2 (LRRK2), ten-
induced kinase 1 (PINK1), arkin (PRKN), and protein deglycase (DJ-1), are found to increase the
susceptibility to PD and have been used to create genetically modified animal models of PD [62,63] . However,
many mouse models do not recapitulate the selective and progressive neurodegeneration seen in PD [64,65] .
Although non-human primate models of PD have been established for investigation [66,67] , it is difficult to
establish a cohort of PD monkey models. Some teams thus explored the generation of pig models to study
the neurological phenotypes of PD.

Yao et al. used TALENs combined with SCNT and embryo transfer to generate DJ-1 KO piglets by
disrupting the PARK7 gene to model the phenotype of PD. Unfortunately, the piglets all died due to cloning
defects, although DJ-1 protein was successfully repressed in all the detected tissues . Another group used
[68]
CRISPR/Cas9 combined with SCNT to generate PARK2 and PINK1 double-gene KO pigs. However, as with
mouse PD models, no phenotypic symptoms of PD were observed in the seven-month-old live mutant
pigs . In 2016, Wang et al. generated a PD pig model using CRISPR/Cas9 system by simultaneously
[69]
targeting three distinct genomic loci, Parkin/DJ-1/PINK1, in Bama miniature pigs. However, the piglets
remained healthy with a normal growth rate, and no typical symptoms of Parkinson’s disease were observed
in the 10-month-old live mutant pigs in this study .
[70]
BASE EDITING USED IN PIG MODELS
Although the CRISPR/Cas9 system has been widely used to facilitate genome editing, it could induce
[35]
random insertions or deletions (indels) through error-prone NHEJ rather than the error-free HDR . As a
result, indels are obtained much more frequently at targeting sites than single-nucleotide substitutions.
However, most human neurological diseases are induced by point mutations, rather than indels , which
[71]
emphasizes the importance of the application of the genome-editing technique of base editing in the
establishment of animal models of human neurological disease.

Base editing is a genome-editing technique that generates mutations at single-base resolution [72-74] . All four
transition mutations, namely C to T, G to A, A to G, and T to C, can be inserted into the genome with the
available CRISPR/Cas base editors (BEs). The cytosine base editor (CBE) can insert a C-G to T-A mutation,
while the adenine base editor (ABE) can alter an A-T base pair into a G-C pair. In RNA, conversion of A to
inosine (I) is also possible with the RNA base editor (RBE) .
[75]