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\begin{document}

\title{A CRISPR-engineered swine model of COL2A1 deficiency recapitulates
altered early skeletal developmental defects in humans}



\author[1]{Boyan Zhang}%
\author[2]{Chenyu Wang}%
\author[2]{Yue Zhang}%
\author[3]{Yuan Jiang}%
\author[1]{Yanguo Qin}%
\author[4]{Daxin Pang}%
\author[1]{Guizhen Zhang}%
\author[1]{He Liu}%
\author[3]{Zicong Xie}%
\author[4]{Hongming Yuan}%
\author[4]{Hongsheng Ouyang}%
\author[1]{Jincheng Wang}%
\author[4]{Xiaochun Tang}%
\affil[1]{Jilin University Second Hospital}%
\affil[2]{Jilin University First Hospital}%
\affil[3]{College of Animal Sciences, Jilin University}%
\affil[4]{Jilin University}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Loss-of-function mutations in the COL2A1 gene were recently described as
a cause of type II collagenopathy, a major subgroup of genetic skeletal
diseases. However, the pathogenic mechanisms associated with COL2A1
mutations remain unclear, and there are few large-mammal models of these
diseases. In this study, we established a swine model carrying COL2A1
mutations using CRISPR/Cas9 and somatic cell nuclear transfer
technologies. Animals mutant for COL2A1 exhibited severe skeletal
dysplasia characterized by shortened long bones, abnormal vertebrae,
depressed nasal bridge, and cleft palate. Importantly, COL2A1 mutant
piglets suffered tracheal collapse, which was almost certainly the cause
of their death shortly after birth. In conclusion, we have demonstrated
for the first time that overt and striking skeletal dysplasia occurring
in human patients can be recapitulated in large transgenic mammals. This
model underscores the importance of employing large animals as models to
investigate the pathogenesis and potential therapeutics of skeletal
diseases.%
\end{abstract}%



\sloppy


\textbf{Keywords}

COL2A1 mutation, CRISPR/Cas9, swine model, genetic skeletal diseases,
cartilage development, tracheal collapse

\textbf{Introduction}

Genetic skeletal disorders (GSDs) arise through mutations that disrupt
normal skeletal development and homeostasis(C. Chen et al., 2016).
Although individually rare, GSDs are of important clinical relevance as
a group owing to their high overall frequency(Kornak \& Mundlos, 2003).
The rapid development of genetic diagnostic technology over the past few
decades has led to the identification of 436 GSDs, which have been
classified into 42 groups(Mortier et al., 2019). The type II collagen
group, one of the major GSD groups identified, consists of 17 clinical
disorders that are associated with mutations in the \emph{COL2A1} gene,
and comprise a spectrum of autosomal-dominant disorders characterized by
skeletal dysplasia(Deng, Huang, \& Yuan, 2016; Kannu, Bateman, \&
Savarirayan, 2012). Type II collagen is the most abundant protein in
cartilage, and, together with additional tissue-specific collagens and
proteoglycans, provides structural integrity to this tissue(Myllyharju
\& Kivirikko, 2004). Type II collagen, a fibrillar collagen, is a
molecule composed of homotrimers of collagen alpha 1 type II
(\selectlanguage{greek}α\selectlanguage{english}1{[}II{]})\textsubscript{3} chains containing characteristic repeating
Gly-X-Y sequences that fold into a triple-helical structure. Although
most \emph{COL2A1} mutations are found in the triple-helical region,
mutations have also been identified in the N- and C-propeptide domains
situated at the amino- and carboxyl-terminal ends of the triple-helical
region(Barat-Houari et al., 2016).

Genetic disorders resulting from \emph{COL2A1} mutations, known as type
II collagenopathies, include a spectrum of highly variable
chondrodysplasias that are characterized according to severity. These
include mild types like avascular necrosis of the femoral head (ANFH)
and Stickler syndrome type I (STL 1); severe types like
spondyloepiphyseal dysplasia congenita (SEDC) and Kniest dysplasia; and
lethal types like achondrogenesis type II (ACG 2) and platyspondylic
skeletal dysplasia, Torrance type (PLSD-T)(Deng et al., 2016). Although
663 independent probands previously reported with 460
different\emph{COL2A1} variants have been identified that may be
associated with different phenotypes(Zhang et al., 2020), the causes of
skeletal deformities and the underlying pathomechanisms remain unclear.
To address this, several genetically modified mouse models have been
established and characterized. For example, pronounced short-limbed
dwarfism was developed in a mouse model with deletion of
\emph{Col2a1}exon 7, as well as in transgenic mice expressing the
\emph{Col2a1}p.G574S mutation(Maddox, Garofalo, Smith, Keene, \& Horton,
1997; Metsaranta et al., 1992). Mild phenotypes, such as the ocular
features found in STL 1, have been observed in
\emph{Col2a1\textsuperscript{+/-}}mice(Kaarniranta et al., 2006).
Moreover, a mouse model of SEDC displaying respiratory failure, short
limb bones, and a short vertebral column has also been
established(Barbieri et al., 2003; Donahue et al., 2003; Macdonald et
al., 2013). The frequently lethal phenotypes associated with ACG 2 and
PLSD-T were also seen in mouse models with p.R789C and p.D1469A
\emph{Col2a1} mutations, respectively(Furuichi et al., 2011; Gaiser et
al., 2002). Although mouse models carrying\emph{Col2a1} mutations show
delayed skeletal development and abnormal cartilage composition and
structure, they lack the overt and striking skeletal dysplasia observed
in larger mammals owing to the limited size of their bones and joints.

Pigs have been widely employed in biological studies owing to their
genetic, anatomical, and physiological similarities with humans(Prather,
Lorson, Ross, Whyte, \& Walters, 2013). Many genetically engineered pig
models have been developed for biomedical research into the underlying
mechanisms of human diseases(Holm, Alstrup, \& Luo, 2016; Yan et al.,
2018). However, relatively few studies investigating GSDs have employed
large mammals, including the pig. The articular structure of the major
joints of pigs is more like that of humans when compared with those of
smaller model organisms. Furthermore, the tools currently available for
genetic manipulation, in combination with somatic cell nuclear transfer
(SCNT) technology, allow for the modification of endogenous pig genes and
the development of a variety of pig models of human diseases(Han et al.,
2017; Yang et al., 2017; Zhou et al., 2015).

In this study, we used CRISPR/Cas 9 to modify the pig \emph{COL2A1} gene
and establish a swine model of a lethal type II collagenopathy that
resembles PLSD-T. The generation of a swine model carrying
\emph{COL2A1}mutations, which displayed typical skeletal deformities and
abnormal cartilage development, demonstrates that large mammals can
recapitulate overt and striking skeletal dysplasia caused by genetic
variants. Importantly, investigation of the trachea of \emph{COL2A1}
mutant piglets provided convincing evidence that tracheal collapse was
the most likely cause of the death of the mutant piglets shortly after
birth. Our findings support that genetically modified swine models are
valuable tools for studying the pathogenesis of GSDs and the associated
mechanisms.

\textbf{Materials and Methods}

\emph{Ethical statement.}

All animal studies were approved by the Animal Welfare and Research
Ethics Committee of Jilin University, and all procedures were conducted
strictly in accordance with the Guide for the Care and Use of Laboratory
Animals. All experimental pigs were obtained from the Huichang Animal
Husbandry Science and Technology Co., Ltd. All animal operations were
performed under anesthesia, and every effort was made to minimize animal
suffering.

\emph{sgRNA design and CRISPR/Cas9 vector construction.}

The targeted sgRNAs in Figure 1C were designed according to a previously
described method (http://crispr.mit.edu/)(Cong et al., 2013). The
sequences of the two sgRNA are provided in Figure 1C. The
pX330U6-Chimeric\_BB-CBh-hSpCas9 plasmid was purchased from Addgene
(\#42230). Two complementary sgRNA oligonucleotides with appropriate
adaptors were synthesized and then annealed in standard Taq buffer (NEB,
Beijing, China). The resultant double-stranded DNA was ligated to the
BbsI sites of the vector backbone to form the targeting plasmid.

\emph{Isolation of porcine fetal fibroblasts and cell culture.}

Porcine fetal fibroblasts were derived from 32-day-old Bama miniature
pig fetuses. Fetuses were disaggregated in culture medium containing 200
U/mL collagenase IV (type IV, 260 U/mg; Gibco, Grand Island, NY, USA),
18\% fetal bovine serum (Gibco), 0.0125 mg/mL DNase I (2,000 U/mg;
Sigma, St. Louis, MO, USA), and 1\% penicillin/streptomycin (Gibco) for
5--6 h. Isolated PFFs were resuspended and cultured in 10-cm Petri
dishes until subconfluence, and then cultured in Dulbecco's modified
Eagle's medium (Gibco) supplemented with 12\% fetal bovine serum.
First-generation cells were frozen in fetal bovine serum containing 10\%
dimethyl sulfoxide.

\emph{Porcine fetal fibroblast transfection and selection.}

Approximately 3 \selectlanguage{ngerman}× 10\textsuperscript{6} PFFs were electroporated with 35
\selectlanguage{greek}µ\selectlanguage{english}g of the Cas9/gRNA targeting vector and ssODN template in 220 \selectlanguage{greek}µ\selectlanguage{english}L of
Opti-MEM (Gibco) using a BTX ECM 2001 system (Harvard Bioscience,
Holliston, MA, USA). The electroporated PFFs were cultured at 37\selectlanguage{ngerman}°C for
72 h and then trypsinized for genomic DNA extraction. Target sequence
regions were amplified by PCR using the following parameters: 94°C for 3
min, followed by 35 cycles of 94°C for 20 s, 60°C for 10 s, and 72°C for
33 s, with a final extension at 68°C for 5 min. For the selection of
single-cell colonies, electroporated PFFs were plated onto 10-cm Petri
dishes after 48--72 h of culture. Single-cell colonies were picked and
cultured in 24-well plates for recovery, while a small part of each
colony was lysed for genotyping. The regions surrounding the intended
point mutations were amplified and then sequenced. Amplicons with
multi-peaks were cloned into the pLB vector (Tiangen, Beijing, China) to
identify the exact sequence of each allele.

\emph{Somatic cell nuclear transfer.}

The SCNT protocol was conducted as described by Lai et al(Fan et al.,
2013). Briefly, cumulus-oocyte complexes were matured at 39°C for 40 h
in maturation medium after removing the follicles. The first polar body
was then aspirated from mature oocytes using a glass pipette, and,
finally, the donor cells were fused with enucleated oocytes using BTX
electrofusion equipment. The reconstructed embryos were then cultured
for nearly 12 h before being transferred into surrogate sows. Pregnancy
was monitored by ultrasonography after 30 days. Piglet DNA was extracted
from the tail and analyzed \emph{via} PCR sequencing. The amplicons were
also ligated to the pLB vector to identify the exact sequence of both
alleles.

\emph{Identification of genetically modified piglets.}

The primers designed for amplification of the \emph{COL2A1} -specific
Cas9/gRNA plasmid are shown in Figure 1C. PCR was performed using the
following parameters: 94°C for 5 min, followed by 40 cycles of 94°C for
30 s, 60°C for 30 s, and 72°C for 33 s, with a final extension of 72°C
for 5 min. Genomic DNA from wild-type Bama miniature pigs was used as
negative control.

\emph{Radiographic analysis.}

Autoradiographs of whole-body skeletons and cartilage of piglets were
taken on radiographic \selectlanguage{english}film (ROTANODE, Tokyo, Japan) using the YEMA
Radiography System equipped with a digital camera (Varian, California,
USA). The image exposure factors were 30 kV 2 mAs.

\emph{Histological and immunohistochemical analysis.}

For histological analyses, femurs, humeri, and tracheas were collected
from \emph{COL2A1} mutant piglets and WT piglets after removing the
surrounding muscles and fascia. Femurs, humeri, and tracheas were fixed
in 4\% paraformaldehyde at 4degC. The bones were subsequently decalcified
in 15\% EDTA for 3 weeks, dehydrated in increasing concentrations of
ethanol (70\% for 7 h, 80\% for 2 h, 96\% for 2 h, and 100\% for 4 h),
washed with xylene, and embedded in paraffin. Then, 5-\selectlanguage{greek}μ\selectlanguage{english}m thick sections
were cut and used for H\&E(Esapa et al., 2012a) and safranin O/fast
green staining as previously described(Y. Chen et al., 2017; Schumacher,
Joiner, Less, Drewry, \& Williams, 2016), and were then analyzed under a
microscope (catalog \#BX43, Olympus, Tokyo, Japan). For
immunohistochemical analysis, sections were incubated with an
anti-\emph{COL2A1} primary antibody (catalog \#BA0533; Boster Biological
Technology, Wuhan, China) in 3\% BSA overnight at 4\selectlanguage{ngerman}°C. After washing
with PBS, the sections were incubated with secondary antibody (catalog
\#BA1039; Boster Biological Technology, Wuhan, China) for 30 min, and
the staining intensity was analyzed by microscopy.

\emph{Western blotting analysis.}

Fresh cartilage and trachea samples were ground and then resolved in
lysis buffer. The resultant protein samples were quantified using a BCA
protein assay kit (Beyotime, Haimen, China). The isolated proteins were
separated by SDS-PAGE and then transferred to nitrocellulose membranes.
The blots were incubated with a primary antibody against type II
collagen (\#BA0533, Boster Biological Technology), washed, and then
incubated with a horseradish peroxidase-labeled secondary antibody
(catalog \#BA1039; Boster Biological Technology). Finally, the blotting
bands were detected with an ECL-Plus western blotting reagent
(Beyotime).

\emph{Real-time RT-PCR.}

Total RNA was extracted from femurs, humeri, and tracheas of newborn WT
and \emph{COL2A1} KO piglets using TRNzol-A+ reagent (Tiangen), treated
with DNase I (Fermentas, Massachusetts, USA), and reverse-transcribed to
cDNA using the BioRT cDNA First-Strand Synthesis Kit (Tiangen). qPCR was
performed using the ABI PRISM 7500 FAST Real-Time PCR System (Applied
Biosystems, Massachusetts, USA), and the 2\selectlanguage{english}\textsuperscript{-ΔΔCt}formula
was used to determine relative gene expression, which was normalized to
the level of \emph{GAPDH} mRNA. All experiments were repeated three
times, and the data are expressed as means \selectlanguage{ngerman}± SEM.

\emph{Off-target analysis.}

To test for off-target mutations in the \emph{COL2A1} mutant piglets,
the top eight POTS were selected for each sgRNA using the CasOFFinder
Tool (http://www.rgenome.net/cas-offinder/new). Amplicons were subjected
to Sanger sequence. The primers used are listed in Table S1.

\emph{Statistical analysis.}

A two-tailed Student's \emph{t} -test was used for comparisons between
groups, and one-way analysis of variance (ANOVA) with
Tukey's\emph{post-hoc} test was used for comparisons between multiple
groups. Signi\selectlanguage{english}ficance compared to untreated controls is denoted with
*\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01, and
***\emph{P}\textless{} 0.001 in the \selectlanguage{english}figures and \selectlanguage{english}figure legends.
Statistical analysis was performed in GraphPad Prism 7.0 (GraphPad
Software, La Jolla, CA, USA).

\textbf{Results}

\emph{Generation of a pig model with type II collagen deficiency}

CRISPR/Cas9 technology is an effective approach for the generation of
double-stranded DNA breaks to facilitate homologous
recombination-mediated modification of target genes. We designed two
guide RNAs (gRNAs) specific for \emph{COL2A1} exon 27 where is highly
conserved among different species (Figure 1A), and one single-stranded
donor oligonucleotide (ssOND) for insertion into the \emph{COL2A1}
locus\emph{via} homology-directed repair (HDR) (Figure 1B and 1C). One
of the target gRNAs was tested to verify that double-stranded DNA breaks
could be effectively generated (Figure 1D). Both the Cas9/gRNA
expression vector and single-stranded oligonucleotide (ssODN) were
transfected into porcine fetal fibroblasts (PFFs). We screened 366 PFFs
by PCR and identified 7 positive clones heterozygous for a mutation in
exon 27 of the \emph{COL2A1} gene. We selected a cell clone that
contained a compound heterozygous mutation and obtained 1301 embryos
following SCNT. One of the compound \emph{COL2A1} variants,
c.1744G\textgreater{}A, was previously reported to be associated with
ANFH(Kishiya, Nakamura, Ohishi, Furukawa, \& Ishibashi, 2014), while a
novel variant, c.1749delA, was predicted to result in a frameshift
mutation (Figure 1E). All the embryos were surgically transferred to
five surrogate sows, three of which became pregnant. Seven piglets were
born by natural delivery. However, the piglets all died shortly after
birth, and all carried the compound heterozygous mutations identified by
PCR, T-cloning and Sanger sequencing. Additionally, the piglets
exhibited many of the phenotypical features associated with type II
collagenopathies and afforded us an opportunity to establish the cause
of the premature death of the animals. As off-target effects are a major
concern when using the CRISPR/Cas9 system, we performed Sanger
sequencing for eight potential off-target sites (POTS). No off-target
effects were found in the\emph{COL2A1} mutant piglets (Supporting Figure
S1 and Supporting Table S1).

\emph{Phenotype characterization of COL2A1 knockout pigs}

As shown in Figure 2A, \emph{COL2A1} knockout (KO) piglets were shorter
than the controls. The flat face, cleft palate, and limb hypoplasia
observed in the KO pig are phenotypes typically seen in patients with
type II collagenopathy (Figure 2B to 2D)(Zhang et al., 2020). The cleft
palate is also considered as a typical phenotype caused by
\emph{COL2A1}variants in mouse models(Barbieri et al., 2003; Vandenberg
et al., 1991). Moreover, the \emph{COL2A1} KO piglets had a short nose
indicative of mid-face hypoplasia, and the ratio of abdomen
circumference to chest circumference suggested that the KO piglets had a
protuberant abdomen (Figure 2E).

Radiographic examinations showed that the \emph{COL2A1} KO piglets had a
markedly underdeveloped skeleton. Lateral radiographic views showed a
depressed nasal bridge and short maxilla, which corresponded to the
visible flat-face phenotype. Ovoid vertebral bodies could also be
observed in the KO pigs (Figure 2F). Radiographic images of
the\emph{COL2A1} KO pigs further showed thoracic dysplasia, shortened
long bones with ragged metaphyses, iliac hypoplasia, and brachydactyly
with short metacarpals (Figure 2G to 2I). Figure 2J shows the measured
length of the forelimb, hindlimb, spine, and maxilla.

\emph{Bone structure is abnormal in COL2A1 KO pigs}

For analysis of bone and cartilage pathology, we isolated the long bones
and ribs of both KO and wild-type (WT) piglets. We found that the long
bones of KO pigs were significantly shorter than those of WT pigs
(Figure 3A and 3B). In the \emph{COL2A1} KO pigs, the heads of the
humerus and femur were underdeveloped, and all the long bone epiphyses
were soft and the margins, especially that of the joint structure, were
unclear. Interestingly, the ribs of \emph{COL2A1} KO pigs were also
shorter than those of WT pigs, which resulted in a narrower thoracic
cavity (Figure 3C). The lengths of the long bones and ribs are shown in
Figure 3D.

\emph{Skeleton dysplasia in COL2A1 KO pigs restricts organ development}

To explore whether the skeleton deformities affected organ development,
we isolated the organs of both \emph{COL2A1} KO and WT pigs (Figure 3E).
In the mutant pigs, the absolute weights of all the organs were
marginally smaller than those of the controls, and showed no significant
differences in morphology (Figure 3E and 3G). However, the weight of the
lungs of \emph{COL2A1} KO pigs was lower than that of WT pigs,
indicating that the deformity of the rib cage restricted lung tissue
development (Figure 3F). During the process of isolating the lung, we
found that the tracheas of KO pigs were soft and inelastic, a critical
factor that prompted us to further explore the cause of death of the
COL2A1 mutant pigs shortly after birth.

\emph{Histomorphology and pathology of the articular cartilage of the
limbs of COL2A1 KO pigs}

To understand the pathogenic basis for the skeletal dysplasia phenotype,
we investigated the histology of the articular cartilage of
the\emph{COL2A1} KO pigs (Figure 4). The cartilage of the KO pigs
presented porous areas, as well as barely defined superficial,
transitional, and deep zones, the three layers that comprise normal
articular cartilage. Compared with that of WT piglets (Figure 4A-\selectlanguage{greek}α, β\selectlanguage{english}),
the femoral head cartilage of KO piglets (Figure 4A-\selectlanguage{greek}γ\selectlanguage{english}) presented
vacuolized chondrocytes (arrows) and disrupted collagen formation.
Similar changes could be seen in the cartilage of knee joint (Figure
4B). Besides, the lack of regular alignment of hypertrophic chondrocytes
was seen in the Figure 4A-\selectlanguage{greek}γ \selectlanguage{english}and Figure 4B-\selectlanguage{greek}γ.

\selectlanguage{english}Articular cartilage formation in the hip and knee joint was assessed by
safranin O/fast green staining (Figure 5A and 5C). Safranin O staining
intensity was greatly reduced in the articular cartilage of\emph{COL2A1}
KO pigs compared with that of WT animals. We also compared the
immunoreactivity of type II collagen in the articular cartilage of WT
and \emph{COL2A1} KO pigs by immunohistochemical analysis using sections
from the femoral head (Figure 5B) and knee articular cartilage (Figure
5D), and found that the expression of type II collagen was decreased in
KO pigs.

The morphological results of hematoxylin and eosin (H\&E), safranin
O/fast green, and immunohistochemical staining of humerus head and
trochlea sections are shown in Supporting Figure S2 and S3. Besides, the
thickness of the cartilage in each joint was compared and exhibit in
Supporting Figure S4.

\emph{Histomorphology and pathology of the tracheal cartilage in COL2A1
KO pigs}

Based on our finding that the tracheas of KO pigs were soft and
inelastic, we further investigated the pathological status of the
tracheal cartilage. As can be seen in H\&E-stained sections, the
tracheal cartilage of \emph{COL2A1} KO pigs was flattened and lacked the
typical C-shaped curve, and the lumen was essentially obliterated (Grade
IV) (Figure 6A). Moreover, the alignment of the pseudostratified
ciliated columnar epithelium was lost, and the goblet cells were larger
than those of WT pigs (Figure 6A-\selectlanguage{greek}γ\selectlanguage{english}). Although the thickness of the
tracheal cartilage was similar between \emph{COL2A1} KO and WT piglets,
the mutant piglets presented a reduced number of chondrocytes (Figure
6B). Immunohistochemical analysis further indicated that the expression
of type II collagen was reduced in the tracheal cartilage of KO pigs
(Figure 6C). The results of the picrosirius red staining demonstrated
that there were fewer collagen fibers in the perichondrium and less
hyaline cartilage in the trachea of KO pigs than in those of WT pigs
(Figure 6D).

Healthy tracheal cartilage helps keep the airways open when breathing;
however, lack of type II collagen expression leads to tracheal collapse
and blockage of fresh air entry into the lungs, which may have been the
cause of the premature death of our \emph{COL2A1} KO piglets.

\emph{Type II collagen was decreased in the articular cartilage of
COL2A1 KO pigs}

To confirm that the loss of \emph{COL2A1} expression in the KO piglets
was indeed due to the mutations generated in the \emph{COL2A1} gene,
western blot and real-time qPCR was performed for the articular
cartilage of the major joints (hip, knee, shoulder, and elbow). As shown
in Figure 7A and 7B, the COL2A1 protein expression level was notably
reduced in KO pigs compared with that of control pigs, while qPCR
analysis indicated that \emph{COL2A1} mRNA levels were significantly
reduced in the articular and tracheal cartilage of KO pigs when compared
with the levels in WT pigs (Figure 7C to 7E). The predicted 3D structure
models suggested that the structure of type II collagen would be
disrupted as a result of the c.1749delA mutation in the
\emph{COL2A1}gene (Figure 7F). The above data indicated that the level
of type II collagen was decreased in the mutant piglets, which resulted
in underdeveloped cartilage and skeletal deformities.

\textbf{Discussion}

In this study, we generated genetically modified swine carrying
\emph{COL2A1} mutations that successfully recapitulated abnormal early
bone and cartilage development in humans. Importantly, our swine model
provided convincing evidence that tracheal collapse was the most likely
cause of the premature death of the \emph{COL2A1} mutant piglets that
occurred shortly after their birth. Additionally, this is the first
genetically modified swine model generated for the investigation of
GSDs, which expands the range of applications of genome editing
technology in large mammals for human disease modeling.

In our study, a swine model harboring compound heterozygous mutations
(c.1744G\textgreater{}A and c.1749delA) in the \emph{COL2A1} gene
displayed phenotypes typically observed in skeletal dysplasia. The
c.1744G\textgreater{}A mutation was previously identified in three
generations of a family that all suffered from bilateral hip joint
lesions and were diagnosed with ANFH(Kishiya et al., 2014). The severe
skeletal defects in our \emph{COL2A1} mutant piglets, such as
significantly shorter limbs, narrower thoracic cavity, and
underdeveloped vertebral body, correspond to the clinical phenotypes of
PLSD-T(Nishimura et al., 2004), a rare skeletal dysplasia characterized
by extremely short limbs, platyspondyly, and severe pelvic
hypoplasia(Nishimura et al., 2004; Zankl et al., 2005). This disorder is
usually caused by mutations in the C-propeptide region of
\emph{COL2A1}and is lethal in the perinatal period in most reported
cases(Desir et al., 2012; Nishimura et al., 2004; Okamoto et al., 2012;
Zankl et al., 2005). Skeletal disorders similar to those of PLSD-T
patients also occurred in a mouse model with an ENU-induced missense
mutation (c.4406A\textgreater{}C) in the C-propeptide coding region
of\emph{Col2a1} (Furuichi et al., 2011). In this mouse model,
heterozygotes were smaller than their WT siblings and showed
disproportionately short limbs and slightly hypoplastic vertebral
bodies, while homozygotes died at birth and exhibited severe dwarfism
with shortened limbs and snout. Other mouse models of GSDs have also
been established, including for SEDC, osteoarthritis, and
disproportionate micromelia(Esapa et al., 2012b; Helminen et al., 1993;
Pace et al., 1997).

Histological analysis showed that the \emph{COL2A1} KO piglets displayed
reduced articular cartilage thickness in the major limb joints compared
with that of WT piglets, and the articular cartilage tissue exhibited
porous areas and indiscernible superficial, transitional, and
proliferative zones. Moreover, the regular alignment of hypertrophic
chondrocytes was lost, as has also been reported for \emph{Col2a1}mutant
mice, although porous areas were not seen in the cartilage tissue of
these animals(Esapa et al., 2012a; Garofalo et al., 1991; Liang et al.,
2014). A previous study suggested that loss of chondrocyte columnar
organization was due to the increased deposition of mutated type II
collagen in cartilage, as was also seen in other transgenic mouse models
of SEDC(Donahue et al., 2003; Gaiser et al., 2002). However, the
mechanism involved in the loss of the hypertrophic zone in cartilage has
also been reported to result from the activation of the endoplasmic
reticulum (ER) stress-unfolding protein response-apoptosis cascade
resulting from the accumulation of mutated type II collagen, that
finally leads to chondrodysplasia(Chung, Jensen, Gawron, Steplewski, \&
Fertala, 2009; Liang et al., 2014). The enlarged and extended
chondrocyte phenotype seen in our model is also typically observed in
affected human patients with \emph{COL2A1} mutations and \emph{Col2a1}
mutant mice(Esapa et al., 2012a; Su et al., 2010). A mutant form of type
II collagen was revealed to lead to articular chondrocyte hypertrophy
through the inhibition of integrin beta 1/SMAD1 interaction(Lian et al.,
2019). Surprisingly, in the safranin O-/fast green-stained sections of
cartilage tissue of the femoral head (Figure 4C), red safranin O
staining, which represents cartilage, was absent. We suspect that this
was due not only to the \emph{COL2A1} mutations leading to reduced
cartilage formation, but also the lack of the type II collagen in the
cartilage. Other safranin O-/fast green-stained sections also showed
reduced safranin O staining intensity (Figure 5A; Supporting Figure S3A
and S3C), which has also been reported in previous studies(Fernandes,
Seegmiller, Nelson, \& Eyre, 2003). Notably, we did not perform
transmission electron microscopy (TEM) analysis as the piglets died too
abruptly to enable fresh tissue collection. However, other studies have
indicated that the rough ER and Golgi bodies in mutated chondrocytes are
dilated, and there is aberrant accumulation of glycogen granules, as
well as fewer and thinner collagen fiber aggregates, in the cartilage
matrix of \emph{Col2a1} mutant mice(Arita et al., 2002; Garofalo et al.,
1991; Li et al., 1995).

Importantly, in our study, we detected tracheal collapse in\emph{COL2A1}
mutant piglets, which was almost certainly the cause of their death
shortly after birth. We found that the tracheal cartilage
of\emph{COL2A1} mutant piglets was flattened, lacking the typical
C-shaped curve, and the lumen was essentially obliterated. The
picrosirius red-stained sections showed a decreased density of collagen
fibers in the mutant piglets (Figure 6D). Additionally, in our
\emph{COL2A1}mutant pigs, the pseudostratified ciliated columnar
epithelium was not aligned, and the goblet cells were larger than those
of control piglets (Figure 6A). These findings have not been previously
reported. Two patients affected with PLSD-T caused by \emph{COL2A1}
mutations were reported to suffer only from respiratory failure in
infancy(Nishimura et al., 2004; Okamoto et al., 2012), while a few
studies have only indicated that transgenic mice with \emph{Col2a1}
mutations show severe respiratory failure(Barbieri et al., 2003; Donahue
et al., 2003). Nevertheless, pulmonary hypoplasia and tracheal collapse
are characteristic findings that have been presumed to be the cause of
neonatal lethality in atelosteogenesis type 1 (AO1), in which some
phenotypes are similar to those observed in PLSD-T(Bejjani et al.,
1998).

The pathological mechanism of \emph{COL2A1} mutation has been study by
lots of research. We have known that \emph{COL2A1} gene encodes the
alpha 1 chain of procollagen type II, and three alpha 1 chains are
folded together in a triple-helical configuration to form the
procollagen homotrimer. After secretion into the extracellular matrix,
the N- and C-terminal propeptides are removed, forming the mature type
II collagen molecule(Bejjani et al., 1998; Strom \& Upholt, 1984).
The\emph{COL2A1} gene comprises 54 exons, with the triple-helical region
ranging from exon 8 to exon 51, flanked by N- and C-propeptide
domains(Barat-Houari et al., 2016). Most mutations in the
\emph{COL2A1}gene occur in the central domain that encodes the
triple-helical region(Hoornaert et al., 2006); consequently, when
mutated, this domain is believed to exert a dominant-negative effect by
interfering with the normal assembly of collagen chains, as shown in a
\emph{Col2a1} mutant (p.R798C) mouse model(Gaiser et al., 2002).
Mutations in exon 2, encoding the N-propeptide domain, cause ocular
disorders such as Stickler syndrome. \emph{COL2A1} mRNA undergoes
alternative splicing, resulting in two isoforms. The long form (type IIA
isoform) is predominantly expressed by chondroprogenitor cells during
early development and in the vitreous of the eye, while the shorter
(IIB) form is expressed by differentiating cartilage chondrocytes in
adults(McAlinden et al., 2008; Richards \& Snead, 2008). Mutations in
exon 2 of \emph{COL2A1} can have dramatically different effects on
pre-mRNA splicing, and lead to Stickler syndrome or other type II
collagenopathies(Richards \& Snead, 2008). Mutations in this exon
usually cause a mild disorder, as demonstrated in a transgenic mouse
model with a mutated 5' splice site of \emph{COL2A1} exon 2; these mice
with apparent expression of only the exon 2-containing IIA mRNA isoform
displayed no overt phenotype and appeared healthy(Lewis et al., 2012).
In contrast, the C-propeptide has been reported to play a crucial role
in the formation of the collagen triple-helical structure(Doege \&
Fessler, 1986; Khoshnoodi, Cartailler, Alvares, Veis, \& Hudson, 2006).
After transfer into the ER, procollagen chains associate \emph{via}their
C-propeptides to form homotrimers, and this interaction is stabilized by
disulfide bonds.

In summary, using a CRISPR/Cas 9 approach, we have established a pig
model carrying compound \emph{COL2A1} mutations. This
\emph{COL2A1}mutant model exhibits overt and typical phenotypes that
resemble those of PLSD-T, and mainly involve abnormal early cartilage
development. Our model paves the way for the establishment of
large-animal models to simulate other hereditary skeletal diseases, such
as ANFH and OA, and to develop effective prevention and treatment
strategies.

\textbf{Acknowledgements}

The authors are grateful to all the animals in this study. And we thank
Jianing Ning, Lin Zhou, Jiahuan Chen, Xiaodong Zou, Lerong Ma and Ang Su
for their excellent technical assistance in this study.

\textbf{Competing interests}

The authors declare no competing interests.

\textbf{Data availability}

All data needed to evaluate the conclusions in the paper are present in
the paper and/or the Supplementary Materials. Additional data related to
this paper may be requested from the authors.

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\textbf{Figure Legends}

\textbf{Figure 1.} Generation of \emph{COL2A1} knockout (KO)
pigs.\textbf{(A)} The target position of amino acid for mutation are
highly conserved among different species. \textbf{(B)} Schematic diagram
of the strategy used to generate \emph{COL2A1} KO pigs
\emph{via}ssODN-mediated homology-directed repair. Two gRNAs were used
to target the pig \emph{COL2A1} exon 27 to promote DNA breaks and
homologous repair. \textbf{(C)} Target sequences of the two sgRNAs and
complementary oligo sgRNAs. \textbf{(D)} Sequencing chromatogram showing
a DNA break in exon 27. The arrow indicates the Cas9 cleavage
site.\textbf{(E)} Sequencing chromatogram showing the compound
heterozygous mutation of one of the cell clones selected for SCNT.

\textbf{Figure 2.} Phenotypic characterization and skeletal radiographs
of the \emph{COL2A1} knockout (KO) piglets. \textbf{(A)} Whole-body
photographs of a symptomatic \emph{COL2A1} KO piglet and a wild-type
(WT) piglet shortly after birth. \textbf{(B)} The underdeveloped nose of
the \emph{COL2A1} KO pig compared with that of the WT pig.
\textbf{(C)}Cleft palate of the KO pig compared with that of the WT
pig.\textbf{(D)} Comparison of the fore- and hindlimbs between KO and WT
piglets. \textbf{(E)} Comparison of average body weight, body length,
length of the nose, and ratio of chest circumference to abdominal
circumference. \textbf{(F)} Autoradiographs showing the abnormal
systemic skeletal development in the \emph{COL2A1} KO pig. A depressed
nasal bridge and short maxilla were noted (yellow arrow). Additionally,
lateral radiographic views showed the presence of ovoid vertebral bodies
in the KO pigs (blue arrow). \textbf{(G)} Thinner and shorter ribs,
smaller and splayed scapulae (yellow arrow), and underdeveloped
vertebrae (blue arrow) in KO piglets compared with the same structures
in WT piglets. \textbf{(H)} The underdeveloped skeleton of the forelimb
in the KO pig compared with that in the WT pig. Shortened long bones
with ragged metaphyses (yellow arrow) and brachydactyly with short
metacarpals (blue arrow) can be observed in the KO pigs. \textbf{(I)}The
underdeveloped skeleton of the hindlimbs of KO piglets compared with
those of WT piglets. Iliac hypoplasia (yellow arrow), ragged metaphyses
(blue arrow), and brachydactyly with short metacarpals (green arrow)
were noted in KO piglets. \textbf{(J)} Comparison of the average length
of the forelimbs, hindlimbs, maxilla, mandible, and spine between WT and
KO piglets shortly after birth. White bar, 2cm. Data are presented as
means \selectlanguage{ngerman}± SEM. *\emph{P} \textless{} 0.05, **\emph{P} \textless{} 0.01,
***\emph{P} \textless{} 0.001.

\textbf{Figure 3.} Abnormal gross morphology of the bones and organs
in\emph{COL2A1} knockout (KO) pigs compared with those of wild-type (WT)
pigs. \textbf{(A)} ~Gross morphology of forelimb bones showing the
greatly reduced lengths of the long bones and unclear margins of the
epiphyses in the KO pigs; humerus, combined ulna and radius, and
forehoof. \textbf{(B)} Gross morphology of hindlimb bones demonstrating
the significantly shorter long bones and deformities in the hindlimbs of
the KO pigs; femur, tibia, and combined fibula and hindhoof.\textbf{(C)}
~Gross morphology of the rib cage of \emph{COL2A1} KO pigs showing a
notable difference in size, indicating that the KO pigs have a narrower
thoracic cavity. \textbf{(D)} ~Comparison of the average length of the
WT and KO piglets. All the bones of the KO pigs were shorter than those
of the WT pigs. \textbf{(E)} ~No obvious differences could be seen in
the sizes and shapes of the organs (liver, heart, spleen, kidney, and
stomach). \textbf{(F)} ~Comparison of the lungs showed that KO pigs
presented smaller lungs and a thinner trachea (arrow). \textbf{(G)} ~The
absolute weights of all the organs were marginally lower in the KO pigs.
Lung volume and weight were significantly decreased in the KO pigs.
White bar, 2cm. Data are presented as means ± SEM. *\emph{P} \textless{}
0.05, **\emph{P} \textless{} 0.01, ***\emph{P} \textless{} 0.001; ns,
not significant.

\textbf{Figure 4.} ~Histomorphology and pathology of hindlimb articular
cartilage in \emph{COL2A1} knockout (KO) pigs. \textbf{(A)} ~Hematoxylin
and eosin (H\&E)-stained sections of the femoral head. The boxed regions
(A-\selectlanguage{greek}α \selectlanguage{english}and A-\selectlanguage{greek}β\selectlanguage{english}) represent the superficial, transitional, and deep zones of
the articular cartilage in wild-type (WT) pigs. The A-\selectlanguage{greek}γ \selectlanguage{english}region
represents the corresponding abnormal cartilage zones of \emph{COL2A1}KO
pigs, characterized by porous areas with enlarged and vacuolized
chondrocytes (arrows). \textbf{(B)} H\&E-stained sections of the knee
joint. The boxed regions (B-\selectlanguage{greek}α \selectlanguage{english}and B-\selectlanguage{greek}β\selectlanguage{english}) represent the superficial,
transitional, and deep zones of the articular cartilage in WT pigs. The
B-\selectlanguage{greek}γ \selectlanguage{english}boxed region shows the disordered cartilage zones of the knee joint
of the KO pig, characterized by porous areas with enlarged and
vacuolized chondrocytes (arrows). Black bar, 500 \selectlanguage{greek}μ\selectlanguage{english}m; red bar, 200 \selectlanguage{greek}μ\selectlanguage{english}m;
green bar, 100 \selectlanguage{greek}μ\selectlanguage{english}m; blue bar, 50 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 5.} Safranin O- and fast green-stained and
immunohistochemical sections of articular cartilage of
hindlimbs.\textbf{(A)} Safranin O- and fast green-stained sections
showing femoral head articular cartilage morphology, demonstrating the
lack of cartilage tissue formation in \emph{COL2A1} KO pigs.
\textbf{(B)}Immunohistochemical staining showing that the expression of
type II collagen was lower in the femoral head of KO pigs than in that
of WT pigs. \textbf{(C)} Safranin O- and fast green-stained sections
showing knee articular cartilage morphology, demonstrating the lack of
cartilage tissue formation in \emph{COL2A1} KO pigs.\textbf{(D)}
~Immunohistochemical staining showing the decreased expression of type
II collagen in the knee joints of KO pigs. Black bar, 500 \selectlanguage{greek}μ\selectlanguage{english}m; red bar,
200 \selectlanguage{greek}μ\selectlanguage{english}m; green bar, 100 \selectlanguage{greek}μ\selectlanguage{english}m; blue bar, 50 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 6.~} Histomorphology and pathology of the tracheal
cartilage in \emph{COL2A1} knockout (KO) pigs. \textbf{(A)} Hematoxylin
and eosin (H\&E)-stained tracheal sections. The results of the H\&E
staining demonstrated that the trachea of the KO pigs had collapsed, and
the lumen was essentially obliterated. The A-\selectlanguage{greek}γ \selectlanguage{english}boxed region shows the
misaligned pseudostratified ciliated columnar epithelium of
the\emph{COL2A1} KO pigs compared with that of WT pigs (A-\selectlanguage{greek}α\selectlanguage{english}).
Furthermore, the A-\selectlanguage{greek}β \selectlanguage{english}boxed region (WT pigs) shows stronger staining of
the tracheal cartilage and a more uniform texture than that of the
cartilage shown in the A-\selectlanguage{greek}δ \selectlanguage{english}region (KO pigs). \textbf{(B)} ~The thickness
of the tracheal cartilage in the KO pigs was comparable with that of WT
pigs; however, there were fewer chondrocytes in the tracheal cartilage
of the KO pigs.\textbf{(C)~} Immunohistochemical staining showing that
type II collagen expression in tracheal cartilage was decreased in KO
pigs compared with that in WT pigs. \textbf{(D)~} Picrosirius red
staining demonstrating that there were fewer collagen fibers in the
perichondrium and less hyaline cartilage in the trachea of KO pigs when
compared with those of WT pigs. Black bar, 500 \selectlanguage{greek}μ\selectlanguage{english}m; red bar, 200 \selectlanguage{greek}μ\selectlanguage{english}m;
green bar, 100 \selectlanguage{greek}μ\selectlanguage{english}m; blue bar, 50 \selectlanguage{greek}μ\selectlanguage{english}m. Data are presented as means \selectlanguage{ngerman}± SEM.
*\emph{P} \textless{} 0.05; **\emph{P} \textless{} 0.01; ***\emph{P}
\textless{} 0.001; ns, not significant.

\textbf{Figure 7.~} Western blot and qPCR analyses of
\emph{COL2A1}expression levels. \textbf{(A)} Western blots of COL2A1 in
cartilage tissues of the hip, knee, and ankle joints from three KO
piglets (F0-1, F0-3, and F0-6). \textbf{(B)} Western blots of COL2A1 in
cartilage tissues of the shoulder, elbow, and wrist joints from three KO
piglets (F0-1, F0-3, and F0-6). \textbf{(C-E)} Relative \emph{COL2A1}
mRNA expression levels in hindlimb joints, forelimb joints, and tracheal
cartilage as quantified by qPCR. \textbf{(F)} Computer modeling of the
COL2A1 3D structure and impact of the \emph{COL2A1} mono-allelic
targeted mutations. Data are presented as means ± SEM.
*\emph{P}\textless{} 0.05; **\emph{P} \textless{} 0.01; ***\emph{P}
\textless{} 0.001.

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