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\begin{document}

\title{Production of MSTN mutated cattle using CRISPR--Cas9}



\author[1]{Gyeong-Mim Gim}%
\author[1]{Dong-Hyuk Kwon}%
\author[1]{Kyeong-Hyeon Eom}%
\author[1]{Joon-Ho Moon}%
\author[1]{Ji-Hyun Park}%
\author[1]{Dae-Jin Jung}%
\author[1]{Dae-Hyun Kim}%
\author[1]{Jun-Koo Yi}%
\author[1]{Jae-Jung Ha}%
\author[1]{Ka-Yeong Lim}%
\author[1]{Jin Su Kim}%
\author[2]{Goo Jang}%
\affil[1]{Affiliation not available}%
\affil[2]{Seoul National University}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Many transgenic animals have been produced using CRISPR--Cas9 technology
to edit specific genes. However, there are few guidelines for the
application of this technique in cattle. The goal of this study was to
produce trait-improved cattle using the genome editing technology
CRISPR--Cas9. Myostatin (MSTN) was selected as a target locus and
synthetic mRNA of sgRNA and Cas9 was microinjected into bovine in vitro
fertilized embryos. As a result, 17 healthy calves were born and 3 of
these showed MSTN mutation rates of 10.5\%, 45.4\%, and 99.9\%,
respectively. Importantly, the offspring with the 99.9\% MSTN mutation
rate had biallelic mutation (-12bp) and a doubling muscle growth
phenotype. In conclusion, we showed that the genome editing technology
CRISPR--Cas9 can produce genetically modified calves with improved
traits.%
\end{abstract}%



\sloppy


\textbf{Production of \emph{MSTN} mutated cattle using CRISPR--Cas9}

Gyeong-Min Gim\textsuperscript{1,2}, Dong-Hyuk
Kwon\textsuperscript{1,2}, Kyeong-Hyeon Eom\textsuperscript{1,2}, JoonHo
Moon\textsuperscript{3}, Ji-Hyun Park\textsuperscript{3}, Won-Wu
Lee\textsuperscript{3}, Dae-Jin Jung\textsuperscript{4}, Dae-Hyun
Kim\textsuperscript{4}, Jun-Koo Yi\textsuperscript{4}, Jae-Jung
Ha\textsuperscript{4}, Ka-Yeong Lim\textsuperscript{5}, Jin-Soo
Kim\textsuperscript{5}, Goo Jang\textsuperscript{1,2}

\textsuperscript{1}Laboratory of Theriogenology and Biotechnology,
Department of Veterinary Clinical Science, College of Veterinary
Medicine and the Research Institute of Veterinary Science, Seoul
National Univeristy,

\textsuperscript{2}BK21 Plus program, College of Veterinary Medicine,
Seoul National University, Seoul, Republic of Korea

\textsuperscript{3}LARTBio Inc., Seoul, Republic of Korea

\textsuperscript{4}Gyeongsangbukdo Livestock Research Institute,
Yeongju, GyeongSang Buk-Do 36052, Republic of Korea

\textsuperscript{5}Center for genome engineering, Institute for Basic
Science (IBS), Seoul 08826, Republic of Korea

\textbf{Correspondence:} Goo Jang, 1 Gwanak-ro, Gwanak-gu, Seoul,
Republic of Korea, 08826

\textbf{E-mail:}\emph{snujang@snu.ac.kr}

\textbf{Tel:} 82-2-880-1280, Fax: 82-2-873-1269

\textbf{Keywords:} Bovine embryos, CRISPR-Cas9, In vitro
fertilization,\emph{MSTN} , Microinjection

\textbf{Abbreviations: MSTN,} myostatin; \textbf{sgRNA,} single guide
RNA; \textbf{ART,} advanced reproductive technologies;
\textbf{SCNT,}somatic cell nuclear transfer; \textbf{T7E1,} t7
endonuclease 1

\textbf{Abstract}

Many transgenic animals have been produced using CRISPR--Cas9 technology
to edit specific genes. However, there are few guidelines for the
application of this technique in cattle. The goal of this study was to
produce trait-improved cattle using the genome editing technology
CRISPR--Cas9. Myostatin (\emph{MSTN} ) was selected as a target locus
and synthetic mRNA of sgRNA and Cas9 was microinjected into bovine in
vitro fertilized embryos. As a result, 17 healthy calves were born and 3
of these showed \emph{MSTN} mutation rates of 10.5\%, 45.4\%, and
99.9\%, respectively. Importantly, the offspring with the 99.9\%
\emph{MSTN}mutation rate had biallelic mutation (-12bp) and a doubling
muscle growth phenotype. In conclusion, we showed that the genome
editing technology CRISPR--Cas9 can produce genetically modified calves
with improved traits.

Many animal product (milk and meat) studies focus on the improvement of
performance traits in cattle because cattle contribute 45\% of the
global animal protein supply for human consumption {[}1,2{]}.
Significant effort has been made to improve the trait of cattle using
advanced reproductive technologies (ART) {[}3,4{]} based on genotyping
and phenotyping analysis. One application of genotyping and phenotyping
breeding is to select and propagate the breeds with high amount of
muscle. Belgian Blue and Piedmontese are the most representative double
muscles cattle breeds. {[}5{]}. Genetic analysis identified the mutation
of \emph{MSTN} (growth and differentiation factor 8) as the causative
factor for enhanced muscle development. Mutations in the gene have also
been observed in the dog {[}6{]}, sheep {[}7{]}, pig {[}8{]} and human
{[}9{]}. However, the incidence of these natural mutations is very low
and selecting and breeding these individuals to establish an independent
breed is time-consuming and costly.

The development of genome editing tools such as zinc finger nucleases
(ZFN), transcription activator-like effector nucleases (TALENs), and
clustered regularly interspaced short palindromic repeats (CRISPR--Cas9)
has provided new and powerful gene editing tools for functional mutation
studies in various biotechnological industries. Applying these genome
editing tools to livestock will contribute to: improvements in cattle
traits, and better understanding of how to prevent and treat cattle
disease {[}10{]}. Proof of concept gene edited cattle---using bovine
somatic cell nuclear transfer (SCNT)---have been produced with enhanced
traits for disease resistance, allergen removal, production, and welfare
{[}11-15{]}. Because SCNT is used to produce cloned offspring with
precise gene edited cells, it is employed in cattle to generate a
valuable model {[}16,17{]}. However, abnormal reprogramming results in
low efficiency and survival rate, limiting the application of SCNT. An
alternative approach to increase the efficiency of gene editing is
through Belgian Blue the microinjection of in vitro fertilized embryos
{[}18{]}. One study used TALENs for editing \emph{MSTN} gene
microinjection of in vitro fertilized cattle embryos {[}19{]}. However,
there are no reports of a live-born genome edited calf using
CRISPR--Cas9 on the \emph{MSTN} locus. Accordingly, the aim of present
study is 1) to develop a method to produce gene edited bovine
pre-implantation embryos using through microinjection with Cas9 mRNA and
2) to produce the \emph{MSTN} edited calves.

\textbf{Materials and methods}

\textbf{Single guide RNA design and testing on bovine fibroblasts}

To produce the \emph{MSTN} mutated cattle, single guide RNA (sgRNA)
for\emph{MSTN} targeting exon2 was designed by CHOPCHOP software
(https://chopchop.cbu.uib.no/) that selects sgRNA candidates for target
genome (Fig 1A-a). Then the sgRNA for \emph{MSTN} , and Cas9 protein
were co-transfected on bovine fibroblast cells. After 3 days of
transfection, they were harvested for extraction of genome DNA. Through
the T7E1 assay, it was confirmed whether the target \emph{MSTN} gene was
mutated.

\textbf{Oocyte in vitro maturation (IVM)}

Ovaries were obtained from a local slaughterhouse and delivered to the
laboratory within 2 hours. The ovaries were aspirated with a 18-gauge
needle to obtain cumulus-oocyte complexes (COCs) from follicles with
2--8~mm in diameter. The COCs with more than three layers of cumulus
cells and evenly distributed cytoplasm were sorted in the study. For
IVM, the COCs were cultured in chemically defined TCM-199 supplemented
with 0.005~IU/mL FSH (Sigma--Aldrich, Cat. no. F2293), 1 \selectlanguage{greek}μ\selectlanguage{english}g/mL
17\selectlanguage{greek}β-\selectlanguage{english}estradiol (Cat. no. E4389), 100 \selectlanguage{greek}μ\selectlanguage{english}M cysteamine (Sigma--Aldrich, Cat.
no. M6500), and 10\% FBS (Gibco, Cat. no. GIB-16000--044) in a
humidified atmosphere of 5\% CO\textsubscript{2} at 38.5 \selectlanguage{ngerman}°C.

\textbf{Sperm preparation, in vitro fertilization (IVF) and in vitro
culture of embryos (IVC)}

Motile spermatozoa were selected by the Percoll gradient method.
Briefly, frozen-thawed cattle semen at 35 °C was filtered by
centrifugation on a Percoll discontinuous gradient (45--90\%) at
1680~rpm for 15 min. To produce the 45\% Percoll solution, 1~mL of
capacitation-TALP medium was added to 1~mL of 90\% Percoll. The sperm
pellet was washed two times by the addition of 3~mL of capacitation-TALP
medium and subsequent centrifugation at 1680~rpm for 5 min. Washed,
motile spermatozoa were used for IVF. Spermatozoa (1--2 ×
10\textsuperscript{6} sperm/mL) were incubated with mature oocytes for
18 h in 50~\selectlanguage{greek}µ\selectlanguage{english}l microdrops of IVF-TALP medium covered with mineral oil
(Nidacon, Cat. no. NO-100) in humidified atmosphere of 5\%
CO\textsubscript{2} at 38.5 \selectlanguage{ngerman}°C. After 18 h of co-incubation, cumulus
cells were removed from presumptive zygotes. The zygotes were cultured
in a two-step chemically defined culture media {[}reference{]} that was
covered in mineral oil in an atmosphere of 5\% O\textsubscript{2}, 5\%
CO\textsubscript{2} and 90\% N\textsubscript{2} at 38.5 °C.

\textbf{Microinjection}

When performing microinjection, Cas9 mRNA (Sigma--Aldrich, Cat. no.
CAS9MRNA) and sgRNA were divided into 4 groups to find the most
appropriate concentration (CB; only TE microinjection, RNA1X; Cas9 mRNA:
100~ng/\selectlanguage{greek}μ\selectlanguage{english}l, sgRNA: 50~ng/\selectlanguage{greek}μ\selectlanguage{english}l, RNA2X; Cas9 mRNA: 200~ng/\selectlanguage{greek}μ\selectlanguage{english}l, sgRNA:
100~ng/\selectlanguage{greek}μ\selectlanguage{english}l, RNA 4X; Cas9 mRNA: 400~ng/\selectlanguage{greek}μ\selectlanguage{english}l, sgRNA: 200~ng/\selectlanguage{greek}μ\selectlanguage{english}l). After 18 h
of IVF, presumptive zygotes were injected with Cas9 mRNA and sgRNA
synthesized by GeneArt Precision gRNA Synthesis Kit (Thermofisher, Cat.
no. A29377) for \emph{MSTN} using a microinjector machine (Eppendorf,
Femtojet\selectlanguage{ngerman}®). The amount of injected Cas9 mRNA and sgRNA for
\emph{MSTN}were different each group (Fig 1 A-b). After 7 days of
microinjection, pre-implantation stage embryos were collected and the
mutation of\emph{MSTN} was observed in vitro or transferred in vivo into
a surrogate cow.

\textbf{Primary cell culture}

Primary cells were obtained by biopsy of the ear skin of calves. The ear
skin was chopped into small pieces with a sterile scalpel and then
washed several times and incubated at 38.5 °C for 4--18 h in Hank's
Balanced Salt Solution (Gibco, Cat. no. 14175095) supplemented with
collagenase (Collagenase type I, Gibco, Cat. no. 17--100--017). The
following day the dispersed cells were washed several times in DMEM
(Gibco, Cat. no. 21068028) and cultured in DMEM supplemented with 10\%
fetal calf serum (Gibco, Cat. no. GIB-11150--059), 1\%
penicillin/streptomycin (Gibco, Cat. no. 15140148), 1\% non-essential
amino acids (Gibco, Cat. no. 11140050), and 100~mM \selectlanguage{greek}β-\selectlanguage{english}mercaptoethanol
(Sigma--Aldrich, Cat. no. M3418).

\textbf{Detection of \emph{MSTN} gene mutation}

Genomic DNA from transgenic primary cells was extracted using a DNA
extraction kit (Qiagen, Cat. no. 69504). The \emph{MSTN} primer was
designed using PRIMER3 software
(\emph{http://bioinfo.ut.ee/primer3-0.4.0/}, Supplementary Table 1), and
the target sequence was amplified by PCR (94 \selectlanguage{ngerman}°C for 5~min, 35--40 cycles
of 94 °C for 20~sec/57 °C for 30~sec/72 °C for 35~sec, and 72 °C for
5~min). The PCR product from each sample was assessed using the T7E1
assay (Toolgene, Cat. no. TGEN\_T7E1) to detect\emph{MSTN} mutations.

\textbf{Gene expression by real-time PCR}

Total RNA was extracted from primary cultured cells using a RNeasy® Mini
Kit (Qiagen, Cat. no. 74106), and complementary DNA was synthesized from
1 \selectlanguage{greek}µ\selectlanguage{english}g of RNA using the RNA to cDNA EcoDry\textsuperscript{TM} Premix
(Takara, Cat. no. 639543). Gene expression assay was conducted using
SYBR Green on QuantStudio 3 (Applied Biosystems, Model no. A28132), and
relative cycle threshold (CT) values were normalized by GAPDH. The
primers used in this study are listed in Supplementary Table 2.

\textbf{Embryo transfer and pregnancy diagnosis}

Blastocysts were stored in PBS supplemented with 20\% FBS. A single
blastocyst was transferred on day 7 (estrus = day 0 = day of fusion) to
the uterine horn of each recipient animal using a non-surgical
transcervical method. Pregnancy detection was performed on day 50 post
estrus using rectal palpation and ultrasonography. Pregnant cattle were
checked by rectal palpation and ultrasonography at regular intervals
thereafter.

\textbf{Targeted deep sequencing}

Target sites were first amplified to a size of \textasciitilde{}500 bp
from extracted genomic DNA using KAPA HiFi HotStart DNA polymerase
(Roche, Cat. no. \#KK2502) according to the manufacturer's protocols.
Then, amplicons were amplified again to a size of \textasciitilde{}230
bp, after which the amplicons were amplified using TruSeq HT dual
index-containing primers to add adaptor and index sequences for Illumina
sequencing platforms to each sample {[}20{]}. Primers used in this study
are listed in Supplementary Table 3. Pooled PCR amplicons were purified
using a PCR purification kit (MGmed) and sequenced on a MiniSeq
(Illumina) with paired-end sequencing systems (2x150 bp). Cas-Analyzer
(http://www.rgenome.net/cas-analyzer/\#!) was used to quantify the indel
frequencies from deep sequencing data {[}20{]}.

\textbf{Analysis of MSTN off-target effect}

The potential off-target effects caused by CRISPR--Cas9 in the
three\emph{MSTN} mutant calves were assessed using Cas-OFFinder software
(\emph{http://www.rgenome.net/cas-offinder/}). This software offers a
fast and versatile algorithm that searches for potential off-target
sites of Cas9 RNA-guided endonucleases. In the\emph{MSTN} target site,
candidates of five loci targeting the whole gene of cows were found by
setting the mismatch number to 3. A primer targeting each sequence
(Supplementary Table 1) was prepared to confirm the off-target effect
through T7E1 assay.

\textbf{Blood analysis}

To evaluate the general health status in calves, 5~mL of whole blood was
collected from the jugular vein for complete blood count (CBC) analysis
(Hemavet 950, Drew Scientific, USA) and serum chemistry analysis
(BS-400, Mindray, China)s. The animals were also monitored by a
veterinarian to assess their general health condition.

\textbf{Statistical analysis}

All data are presented as mean \selectlanguage{ngerman}± SEM. Newman--Keuls Multiple Comparison
Test and Tukey's test in a one-way ANOVA were used to evaluate
statistically evaluate differences in embryo development, frequency of
mutagenesis, and mRNA expression level. A value of P\textless{}0.05 was
considered statistically significant. All analyses were performed using
the GraphPad Prism program (version 5.01).



\textbf{Results}

\textbf{Validating single guide RNA for \emph{MSTN} in bovine fibroblast
cells}

In bovine fibroblast cells, the selected sgRNA caused a mutation on the
target site. The mutation on \emph{MSTN} was detected by T7E1 assay
(data not shown) and sanger sequencing. Though the sanger sequencing
results, various types of mutation pattern were observed (Fig 1B). These
results show that the selected sgRNA worked well on the
\emph{MSTN}target site.

\textbf{Developmental competence and mutation efficiency on
microinjection mediated gene editing}

To determine optimal conditions, including Cas9 mRNA, and sgRNA
concentrations for producing mutations in bovine blastocysts, four
experimental conditions were used, then developmental competence and
mutation efficiency were investigated in the blastocyst stage following
each condition (Fig 1C, D, and E). There was no significant difference
in cleavage rate across all 4 groups (data not shown). However, in the
RNA4X group a diminished blastocyst formation rate was observed in
comparison to the control group (Fig 1D). In terms of mutation rate, the
RNA2X group (81.3 ± 17.2\%) had the highest mutation compared the other
3 groups (WT: 0\%, RNA1X: 33.3 ± 16.0\%, RNA4X: 50.0 ± 28.5\%) (Fig 1E).
Thus, the RNA2X condition was selected as the optimal condition for the
further production of \emph{MSTN} mutated cattle.

\textbf{Production of \emph{MSTN} mutated Korean beef cow}

The aim of this study was to produce \emph{MSTN} mutated Korean beef
cattle. Microinjection was performed on fertilized embryos under RNA2X
conditions and cultured blastocysts were used for embryo transfer. The
experiment was repeated four times and 595 oocytes were used to generate
mutant embryos. The cleavage rate was 59.0 ± 21.0\% and 86 blastocysts
(14.5 ± 14.0\%) were produced. A total of 26 blastocysts with high
quality morphology were transferred to surrogate mothers (one blastocyst
per recipient). Some of leftover blastocyst (n = 28) were used to assess
the mutation occurrence on \emph{MSNT} and it was determined to be 71.6
± 44.3\%.

After embryo transfer, 19 of the 26 surrogates were pregnant and 2 of
the 19 fetuses were absorbed in the middle of pregnancy. A total of 17
calves (I.D.: \#1 \textasciitilde{} \#17) were live-born and one calf
has a stillbirth (\#14) from dystocia. Deep sequencing analysis showed
mutations in 3 out of the 17 animals (Fig. 2A). Their
\emph{MSTN}mutation rates were 10.5, 45.4, and 99.9\% for \#6, \#14, and
\#17, respectively. In addition, \emph{MSTN} mutation was detected in
\#17 by T7E1 assay and there were no off-target effects (Fig. 2C).
Real-time PCR was conducted to assess the \emph{MSTN} RNA level of
primary cells from \#17 and \#14. In both individuals the level of
\emph{MSTN} RNA was significantly decreased with wild type (Fig. 2D).
Especially in \#17 with the -12 deletion, enhanced muscle growth was
observed compared with wild type calf (Fig. 2E). There were no abnormal
values in a blood test performed to evaluate the general health status
of 5 offspring (\#4, \#6, \#7, \#9 and \#17) including the knockout one
(\#17) at 8-months-old (Table 1).



\textbf{Discussion}

CRISPR--Cas9 mediated genome editing is a powerful biological technology
that has widespread application. Its application to livestock has been
slow {[}21{]}. In this study, by applying genome editing without the
integration of the transgene, an effective knockout condition was
established at the embryo level and live-born edited offspring were
produced. The \emph{MSTN} knockout was used for proof of concept because
of its clear double-muscling phenotype.

The \emph{MSTN} gene consists of three domains (a signal sequence, a
pro-peptide, and a mature-region). After transcription, translation, and
two cleavage events (pro-peptide convertase by furin and tolloid
protease by BMP-1 metalloprotease), the released mature MSTN protein
dimer regulates the inhibition of skeletal muscle growth {[}22{]}. In
more detail, the first cleavage occurs at the 266th position by Furin,
followed by cleavage at 76th by BMP-1/Tolloid metalloproteinase, and
finally, the released active MSTN protein dimer binds to the receptor
(ActRIIB), resulted in inhibition of muscle growth {[}22-24{]}. Two
representative cattle breeds, Belgian Blue, and Piedmontese, show
natural mutations in this gene, 11 bps deletion, and one base mutation
on mature \emph{MSTN} domain, respectively. These cattle breeds
phenotypically indicate that mature \emph{MSTN} domain mutations
contribute to muscle growth {[}5{]}. To mimic or reproduce those natural
mutations using genome editing technologies, in a previous study, ZFNs
disrupted exon1 locus (Signal Sequences region), subsequently, the
mature \emph{MSTN} domain was broken {[}25{]}. In another study, the
mature domain locus via TALEN was directly targeted and mutated in
microinjected embryos {[}19{]}. In both studies, the phenotype was
observed after mutation of the mature locus of \emph{MSTN} .

In our study it was assumed that disruption of the mature
\emph{MSTN}locus might occur by applying effective sgRNA on the
pro-peptide locus using CRISPR-Cas9. Microinjected embryos were
transplanted, and the muscle outgrowing phenotype was observed in one
calf (\#17). Thus, we thought that gene editing on the pro-peptide locus
region worked well and predicted that the sequence of mature domain
locus might be mutated by CRISPR-Cas9. However, one interesting finding
was observed as a result of sequencing. This was an in-frameshift
(-12bps deletion) knockout in the target locus that did not disrupt the
mature \emph{MSTN}domain region or amino acids of two cleavage regions.
In other words, the 266\textsuperscript{th}-, and
76\textsuperscript{th}- amino acids for furin and proteinase were
respectively conserved. Thus, the active MSTN protein dimer may be
formed and muscle production is suppressed normally, and finally a wild
type offspring should be born. Interestingly, the typical phenotyping
(muscle outgrowth) was observed in \#17 calf and the expression of MSTN
mRNA was decreased (Figure 2). Because there have been no reports of
phenotyping because of this type of mutation, it is hard to explain why
this phenomenon occurred. One possibility is that the 156--160 position
can be thought of as another molecular biological function in addition
to the previously known two cleavage events. Similarly, in-frameshift
mutation of the \emph{MSTN}pro-peptide in mice showed a muscle gain
phenotype {[}26{]}. Importantly, the blood test results of the mutated
calves were normal (Table 1) and the calves showed no issue in their
general health. In the future, we will monitor the growth, including
germline transmission, and investigate how this mutation may have
affected the function of\emph{MSTN} .

Microinjection commonly results in mosaic F0 founder animals that are
then screened for the exact knockout/knockin in the F1 generation
following subsequent breeding. This technique is very effective in
rodent experiments but is not suited to cattle because of their long
gestational periods and single pregnancies. A cattle F0 and F1 system
would take more than 3 years and require high costs. Consequently, most
genome edited cattle are produced using a SCNT approach. However, live,
healthy calf offspring are limited when SCNT is employed because of
abnormal reprograming during embryogenesis. In our study, microinjection
was used to produce live, healthy genome edited calves. Randomly
selected blastocysts were analyzed in vitro by sgRNA/Cas9 mRNA and a
81.3 ± 17.2\% knockout efficiency rate was found. Embryo transfer was
performed and a lower \emph{MSTN} mutant cow generation rate of 17.6\%
was found in vivo. It is possible that non-mutated blastocysts were
selected during the randomly selected process. In the future, to improve
the efficiency of producing mutated offspring a portion of the
blastocysts could be biopsied prior to transfer to identify possible
mutations {[}27{]}.

Genotyping analysis showed another interesting result. When mRNA of
sgRNA and Cas9 was introduced into cells and embryos (blastocysts)
various mutant pattern (-12, -10, -3, -2, -1, +1; Figure 1B) were shown,
but only one mutant pattern (-12bps) was observed in genome edited
calves. It is difficult to explain why only one pattern is observed in
all \emph{MSTN} mutated calves. One possible theory is that the cells
with the other mutated pattern may be embryonic lethal at some time
after the point of embryo transfer. Future studies will focus on
improving in vitro and in vivo efficiency.

In conclusion, we demonstrated, for the first time, that microinjection
of Cas9 mRNA and sgRNA for \emph{MSTN} into in vitro fertilized embryos
can produce live, genome edited, Korean beef calves---including one calf
with biallelic mutation. These calves will be served as a model for the
future development of CRISPR--Cas9 technology in the agricultural
industries.

\textbf{Acknowledgments}

This study was financially supported by the National Research Foundation
of Korea (2017R1A2B3004972, NRF-2020R1F1052024), the Research Institute
of Veterinary Science and the BK21 PLUS Program for Creative Veterinary
Science Research, and a SNU-Grant (\#550--2020005). We also thank
members of the G. Jang lab for their valuable comments and the technical
support of SR Han and SY Yum

\textbf{Conflict of interest}

The authors declare no commercial or financial conflict of interest.

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Table 1. Blood analysis in the offspring\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Blood items & References & Offspring & Offspring & Offspring & Offspring
& Offspring\tabularnewline
\midrule
\endhead
& & \#4 & \#6 & \#7 & \#9 & \#17\tabularnewline
WBC & 4.0 \textasciitilde{}
12.0(10\textsuperscript{3}/mm\textsuperscript{3}) & 7.02 & 8.80 & 8.16 &
10.36 & 8.20\tabularnewline
Neutrophil & 0.6 \textasciitilde{}
4.1(10\textsuperscript{3}/mm\textsuperscript{3}) & 3.12 & 4.36 & 3.55 &
5.79 & 3.66\tabularnewline
Lymphocyte & 2.5 \textasciitilde{}
7.5(10\textsuperscript{3}/mm\textsuperscript{3}) & 2.42 & 2.53 & 2.65 &
3.48 & 3.30\tabularnewline
RBC & 5.0 \textasciitilde{}
10.0(10\textsuperscript{3}/mm\textsuperscript{3}) & 8.14 & 10.09 & 9.56
& 8.83 & 8.62\tabularnewline
HGB & 8.0 \textasciitilde{} 15.0 (g/dL) & 11.20 & 12.80 & 11.60 & 11.20
& 10.70\tabularnewline
HCT & 24.0 \textasciitilde{} 46.0 (\%) & 26.30 & 29.30 & 27.70 & 25.10 &
27.20\tabularnewline
PLT & 200 \textasciitilde{}
800(10\textsuperscript{3}/mm\textsuperscript{3}) & 441.00 & 286.00 &
431.00 & 352.00 & 395.00\tabularnewline
AST & 53 \textasciitilde{} 162 & 55.00 & 78.00 & 63.00 & 75.00 &
68.00\tabularnewline
ALP & 77\textasciitilde{} 265 & 209.00 & 321.00 & 331.00 & 390.00 &
343.00\tabularnewline
BUN & 10 \textasciitilde{} 25 & 21.00 & 12.00 & 13.00 & 12.00 &
12.00\tabularnewline
Creatinine & 0.4 \textasciitilde{} 2.0 & 0.90 & 1.10 & 0.90 & 1.00 &
1.70\tabularnewline
Total Protein & 7.2 \textasciitilde{} 9.0 & 7.40 & 8.30 & 8.20 & 7.90 &
7.60\tabularnewline
Albumin & 3.2 \textasciitilde{} 4.2 & 3.80 & 4.20 & 4.10 & 4.00 &
4.10\tabularnewline
Ca (mg/dl) & 8.3 \textasciitilde{} 10.4 & 9.40 & 9.55 & 9.77 & 9.35 &
9.99\tabularnewline
P (mg/dl) & 4.2 \textasciitilde{} 7.7 & 9.02 & 7.76 & 9.64 & 9.22 &
9.23\tabularnewline
Total Cholesterol & 73 \textasciitilde{} 280 & 144.00 & 112.00 & 110.00
& 104.00 & 152.00\tabularnewline
Glucose & 31 \textasciitilde{} 77 & 78.00 & 84.00 & 90.00 & 75.00 &
87.00\tabularnewline
Triglyceride & 7 \textasciitilde{} 323 & 15.00 & 10.00 & 14.00 & 12.00 &
30.00\tabularnewline
Total Bilirubin & 0.01 \textasciitilde{} 0.5 & 0.029 & 0.026 & 0.031 &
0.023 & 0.028\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure legends}

Figure 1. Efficient production of \emph{MSTN} mutant blastocysts. A)
Schematic design for \emph{MSTN} target site (a) and graphical
illustration on analyzing microinjected embryos (b) The red letters and
line represent \emph{MSTN} target site, and the green letters represent
PAM sequence. B) \emph{MSTN} mutation sequence pattern on pooled cells
after transfection with sgRNA and Cas9 protein. C) Representative
pictures of bovine blastocyst on day 7 for each experiment condition
after microinjection (WT: microinjection with Tris-EDTA buffer, RNA1X;
Cas9 mRNA: 100~ng/\selectlanguage{greek}μ\selectlanguage{english}l, sgRNA: 50~ng/\selectlanguage{greek}μ\selectlanguage{english}l, RNA2X; Cas9 mRNA: 200~ng/\selectlanguage{greek}μ\selectlanguage{english}l,
sgRNA: 100~ng/\selectlanguage{greek}μ\selectlanguage{english}l, RNA 4X; Cas9 mRNA : 400~ng/\selectlanguage{greek}μ\selectlanguage{english}l, sgRNA: 200~ng/\selectlanguage{greek}μ\selectlanguage{english}l); D).
The blastocyst formation rate after microinjection with each condition.
*P \textless{} 0.05, **P \textless{} 0.01, ***P \textless{} 0.001.
E)\emph{MSTN} mutation rate of day 7 blastocysts for each condition.

Figure 2. Birth of \emph{MSTN} mutated Korean beef calves. A) Mutation
rate of fetuses (labeled \#1--17) on \emph{MSTN} target site by deep
sequencing. B) Mutation assay using T7E1 assay for one calf (\#17) (WT:
wild type, NC: negative control, PC: positive control, W- :T7E1 assay
without wild type genomic DNA, W+: T7E1 assay with wild type genomic
DNA). C) Analysis of off-target effects on five candidate genes. D)
Relative expression of MSTN mRNA. The bar graph represents the fold
changes in mRNA levels, and the error bars show SEMs (n=3). *P
\textless{} 0.05, **P \textless{} 0.01, ***P \textless{} 0.001. E)
Representative pictures of a biallelic knockout calf (\#17) along with
age (a: 1 month, b: 2 months, c: 3 months, and d: 4 months).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}

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