CHARACTERIZATION OF CRISPR/CAS9-MEDIATED MYOSTATIN KNOCKOUT QUAIL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1019637
• Grant No.: 2020-67030-31338
• Cumulative Award Amt.: $200,000.00
• Proposal No.: 2018-09301
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2023
• Grant Year: 2020
• Program Code: [A1801]- Exploratory: Exploratory Research

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Animal Sciences

Non Technical Summary
A greater understanding of mechanisms regulating muscle growth and development is essential to improve meat production in domestic animals. Muscle growth results from increases in numbers and/or size of muscle fibers. Myostatin (MSTN) inhibits muscle growth and natural or induced mutations of the MSTN gene increases muscle growth in mammals and fish. However, mechanisms (fiber number/size) of enhancing muscle growth by inactivation of the MSTN gene are different and degrees of the differences vary among different species. So far, any avian model with an inactive MSTN gene has not been developed. We recently developed a quail line with an inactive MSTN gene. The proposed research is focused on characterization of pro-myogenic activity of MSTN inactivation in the quail model. In mammals, increased total myofiber numbers (hyperplasia) are determined during fetal development with a continuous maternal supply of nutrients and energy. However, avian embryogenesis occurs independent of the hens in an egg consisting of a fixed size and containing a limited supply of nutrients. Therefore, we will also investigate the interaction of these egg environmental factors and genetics on the resulting phenotype of MSTN inactivation in the avian model. This exploratory grant will generate valuable information that will be a foundation for a future study focusing on 1) determining the critical time window of MSTN function so as to develop approaches to inactivate MSTN at specific permissive stages of muscle development or ages, 2) studying comparative aspects in molecular and cellular mechanisms, and 3) extending this proof-of concept in other major poultry species.

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3299	1050	50%
305	3299	1020	30%
303	3299	1030	10%
305	3299	1040	10%

Knowledge Area
303 - Genetic Improvement of Animals; 305 - Animal Physiological Processes;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1020 - Physiology; 1050 - Developmental biology;

Keywords

crispr/cas9

knockout

muscle

myostatin

poultry

quail

Goals / Objectives
1) Generation of MSTN+/+, +/-, -/- quail by breeding MSTN+/- quail breeders2) Characterization of growth performance with proportional muscle weight3) Measurement of total fiber numbers and fiber size4) Investigation of relationship between egg sizes and chick phenotypes including body weights and muscle phenotypes across MSTN+/+, +/-, -/- lines.

Project Methods
1. Male and female G1 MSTN+/− quail will be bred to generate G2 quail with three different MSTN genotypes (MSTN+/+, +/−,−/−) which will be determined by a PCR-RFLP with Rsa1 restriction enzyme that cut GTAC sequences where GT was deleted in the KO quail.2. A piece of each muscle will be stored at -80°C to validate MSTN KO by Western blot analysis using avian-specific MSTN N-terminal antibodies.3. The pectoralis major muscle and gastrocnemius muscle will be fixed in 10% formalin for an analysis of histomorphometricparameters including fiber sizes and numbers. Muscle sections 6 μm in length will be stained with hematoxylin and eosin using standard histological techniques. The diameter of each fiber will be measured as the shortest distance through the midpoint of all complete fibers in the field (500 fibers per individual) using Image-Pro Plus software (Media Cybernetics, Bethesda, MD).4.To investigate relationship between egg sizes and chick phenotypes including body weights across MSTN+/+, +/-, -/- quail, male and female MSTN +/- will be bred to generate eggs. Based on the size distribution of eggs (n=150) from the original population of quail, the top 33% weighed more than 9 g, medium size (2nd 33%) ranging from 8.35-9.0 g, and the lowest 33% below 8.35 g. There will be a 3x3 factorial design: 3 genotypes X 3 sizes of eggs, a total of 9 groups (n=20 per group). In this experiment, females will be excluded because of variation of BW during the egg laying period. BW (n=20) will be measured weekly from hatch. At age of 77 days, quail (10 quail per group) will be euthanized to measure final BW, and collect the pectoralis major muscle and gastrocnemius muscle. The muscle will be analyzed for histomorphometric parameters including fiber sizes and numbers.

Progress 06/01/20 to 05/31/23

Outputs
Target Audience:Target audiences will be researchers in the area of animal science, developmental biology, poultry science, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the pharmaceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 2 postdoc, 1 technician, and 3 undergraduate student were trained to learn concepts of genome editing and assay skills and tasks for phenotyping. The genome editing concept they learned included embryo handing, micro injection, development of embryos into adult birds, and breeding of chimeras. The assay skills include poultry handling, tissue sampling, quantitative real-time PCR, Western blot, and analysis of histology of muscle tissues. In addition, researchers learned how to interpret Scanning Electron Microscope images of eggshell and bone parameters obtained fromMicro-Computed Tomography scanning. The project provided opportunities to the researchers to learn muscle biology, developmental biology, molecular biology, and reproduction by investigating effects of MSTN mutation on physiological changes in quail. By understanding the physiological differences by genetic mutation, researchers also grasp the importance of integration of multiple research areas in advancing our knowledge in improving the poultry industry. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the project were published in scientific journals to be available to the scientific community in animal and poultry sciences. The results from the project were also presented in the virtual or in-person scientific meetings at the Ohio Agricultural Research and Development Center and Poultry Science Association. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Myostatin (MSTN) negatively regulates in muscle growth and development. For this project, MSTN mutation was generated using the CRISPR/Cas9 system in quail to investigate MSTN function. Body weights of MSTN homozygous mutant (MSTN-/-) quail were significantly heavier compared to wild-type (MSTN+/+) quail. In addition, MSTN-/- showed heavier muscle mass compared to MSTN+/+. Especially, increased total fiber number of pectoralis major and gastrocnemius muscles without change in the muscle fiber size showed that muscle fiber hyperplasia in skeletal muscle resulted in increased body weight and muscle mass of MSTN-/- quail. 2. To investigate feed efficiency, food conversion ration (FCR) was calculated by measuring body weight gain (WG) and feed intake (FI) from post-hatching day (D) 10 to D20, from D20 to D30 and from D30 to D40. FCR from D30 to D40 and overall period (from D10 to D40) were significantly lower in MSTN-/- compared to MSTN +/+ indicating improved feed efficiency by MSTN mutation in both the male and female. 3. In terms of egg laying characteristics, MSTN-/- laid significantly heavier eggs compared to MSTN+/+. However, onset of egg laying was delayed, and eggs numbers were reduced. These data about MSTN function in growth performance and egg production of quail provide a scientific basis for potential usage of MSTN expression as a selection marker in poultry industries. 4. In this study, several parameters for eggshell quality, including eggshell size, weight, breaking strength (EBS), and thickness, were compared. MSTN mutant eggs had greater height and width along with heavier eggshell weight compared to WT eggs, which shows proportional improvement in egg size as affected by the MSTN mutation. However, EBS and eggshell thickness were decreased in mutant eggs compared to WT eggs. In addition, the palisade layer was also decreased without a change in the number of vesicular holes. These data indicated that decreases in the thickness of the eggshell and the palisade layer would be a main factor contributing to a lower EBS in mutant eggs. 5. Tibia bones were collected from MSTN mutant and WT male quail at 4months of age and analyzed by Micro-Computed Tomography scanning. We found positive effects of MSTN mutation on tibia bone sizes and strength, and mineralization. Especially, MSTN mutant resulted in improvements in bone qualities and structural soundness of both diaphysis and metaphysis regions with significant changes in trabecular bones. Taken together, MSTN can be considered as a potential target to improve bone qualities that can reduce the incidence of leg bone problems for the broiler industry. 6. The function of the MSTN gene on regulation of bone quality in layers was also investigated. Tibia bones were collected from WT and MSTN mutant female quail at 5 wk old and 4 mo old, representing prelaying and actively laying stages, respectively. At 5 wk of age, MSTN mutant female quail showed higher bone breaking strength (BBS) and values on parameters related to bone quality such as bone mineral contents (BMC), bone mineral density (BMD), bone volume (BV), and/or trabecular bone thickness in whole diaphysis, whole metaphysis, and metaphyseal trabecular bone, compared to WT female quail. The improved tibia bone quality by MSTN mutation before sexual maturation lasted to a certain degree even after sexual maturation. 7. In avian species, positive relationships between egg weight (EW) and body weight (BW) have been reported. It was reported that EW produced from Mstn homozygous mutant quail (HO) was heavier compared to those from WT quail. In the current study, distributions of pre-incubated EW and associations between EW and BW were compared between the Mstn mutant and WT quail lines. Average egg weight for the HO group was significantly heavier than the WT and the number of eggs having heavier EW (over 11 g) was higher in the HO compared to the WT. BWs at wk (W) 0, 4, and 6 after hatch were also significantly greater in the HO. In addition, linear regression analyses revealed positive relationships between EW and BW from W0 to W6, regardless of sexes and genotypes. Furthermore, Mstn mutant quail were a heavier BW compared to the WT quail originated from eggs with similar weights. These data indicate that increased BW by Mstn mutation is contributed by increased EW and/or growth promoting activity of Mstn mutation independent of increasing egg sizes. 8. We also compared the carcass, meat quality, and histochemical characteristics of pectoralis major (PM) muscle between WT and myostatin homozygous mutant (HO) quail lines. The HO quail line exhibited significantly heavier body weight and PM muscle weight compared to the WT. However, the two groups had similar traits (pH, redness, yellowness, and drip loss) for meat quality. For histochemical traits of PM muscle, Mstn mutant quail exhibited lower type IIA and higher type IIB percentage in the deep region than WT quail, indicating a fiber conversion from the type IIA to IIB. However, the two quail lines had comparable histochemical traits in the superficial region. These data suggest that Mstn mutation greatly increases muscle mass without significantly affecting meat quality. 9. Among different eggshell layers, the cuticle layer is an outermost layer and plays a critical role in protection against bacterial infection. However, the detailed nanostructure of the cuticle layer in the major poultry species has not been investigated. In the current study, eggs from quail, chickens, turkeys, and ducks were collected to image the cross-sectional cuticle layer using a scanning electron microscope (SEM). Unlike the cuticle layer in ducks showing deformed bunched nanospheres, clearly separated nanospheres were present throughout the cuticle layer in quail, chickens, and turkeys. The average size of the nanosphere was the biggest in turkeys and similar between quail and chickens. Most importantly, the size of nanospheres was increased as they ascended from the bottom of the cuticle layer in quail. However, different sizes of nanospheres were randomly distributed throughout the cuticle layer in chickens and turkeys. These new findings in different nanostructures of the cuticle layers in quail, chickens, turkeys, and ducks will serve as a new foundation to better relate their structures with functions. 10. To analyze nanostructure of the cuticle layer of the MSTN mutant and wild-type (WT) quail eggs, SEM images was taken. The size of the nanospheres in the surface of the cuticle layer was increased by MSTN mutation. In addition, increased size of the nanospheres in the MSTN mutant group was also shown in the upper region of the cross-sectional cuticle layer, but not in the lower region of the cuticle layer. However, the number of Escherichia coli attached to the surface did not differ between the two groups. 11. Reverse sexual dimorphism is evident in quail as female quail have a larger body size than males. To understand what characteristics in muscle growth play roles in reversed sexual dimorphism in quail, the weights and the characteristics of the pectoralis major and gastrocnemius muscles (PM and GM, respectively) were compared. Female quail have significantly heavier bodies, and PM and GM weights compared to male quail (1.27-folds, 1.29-folds, and 1.16-folds, respectively). In both PM and GM, there were no differences in total numbers of myofibers and muscle bundles as well as the average numbers of myofibers per bundle between sexes. However, the sizes of myofiber and the bundle were significantly increased in female compared to male (1.33-folds and 1.28-folds in PM, and both 1.23-folds in GM, respectively). The findings of the current study suggest that muscle hypertrophy in female quail, not hyperplasia, can be attributed to the sexual dimorphism in quail size.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lee J, Kim D-H, Lee K. Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide. Int. J. Mol. Sci. 2020; 21:1504.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lee J, Kim D-H, Brower AM, Schlachter I, Lee K. Research Note: Improved feed efficiency in quail with targeted genome editing in the myostatin gene. Poult Sci. 2021;100:101257.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lee J, Kim DH, Brower AM, Schlachter I, Lee K. Effects of myostatin mutation on onset of laying, egg production, fertility, and hatchability. Animals. 2021;11:17.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Lee J, McCurdy C, Chae C, Hwang J, Karolak MC, Kim DH, Baird CL, Bohrer BM, Lee K. Myostatin mutation in japanese quail increased egg size but reduced eggshell thickness and strength. Animals. 2022;12.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lee J, Tompkins Y, Kim D-H, Kim WK, Lee K. The effects of myostatin mutation on the tibia bone quality in female Japanese quail before and after sexual maturation. Poult Sci. 2023;102:102734.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lee J, Tompkins Y, Kim D-H, Kim WK, Lee K. Increased sizes and improved qualities of tibia bones by myostatin mutation in Japanese quail. Front Physiol. 2023;13:18.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Kim DH, Lee J, Choi YM, Lee K. Research Note: Growth promoting potential in Mstn mutant quail dependent and independent of increased egg size. Poult Sci. 2023;102:102260.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Kim D-H, Lee B, Lee J, Bohrer BM, Choi YM, Lee K. Effects of a myostatin mutation in Japanese quail (Coturnix japonica) on the physicochemical and histochemical characteristics of the pectoralis major muscle. Front Physiol. 2023;14:16.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lee J, Choi W, Lee K. Distribution of nanospheres in the cuticle layer of the eggshell in major poultry species. Poult Sci. 2023;102:102882.
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Lee J, Choi W, Kim DH, McCurdy C, Chae C, Hwang J, Kim WK, Lee K. Increased nanosphere size in the cuticle layer of Japanese quail egg by mutation in the myostatin gene. Poult Sci.
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Kim D-H, Lee B, Lee J, Lee K. Muscle Hypertrophy is Associated with Reversed Sexual Dimorphism in Body Size of Quail. Front Zool.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, biotechnology, andindustryNewinformation in this project will provide scientific knowledge for education and a potential application for animal production and the food animal industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 1 postdoc, and 2 undergraduate students were trained to learn concepts of genome editing in poultry and assay skills and tasks for measuring phenotypical changes by MSTN mutation. The assay skills include poultry handling, tissue sampling, and analysis of characteristics of the egg and eggshell. The project provided opportunities to the researchers to learn muscle biology, developmental biology, molecular biology, and reproduction by investigating effects of MSTN mutation on physiological changes in quail. By understanding the physiological differences by genetic mutation, researchers also grasp the importance of integration of multiple research areas in advancing our knowledge in improving the poultry industry. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the project were published in scientific journals to be available to the scientific community in animal and poultry sciences. Students and a postdoc presented the results from the project in the virtual scientific meetings at the Ohio Agricultural Research and Development Center and local meetings. PD also presented in several universities and research institutes. What do you plan to do during the next reporting period to accomplish the goals?For the last year of the project, we will focus on effect of MSTN mutation on myofiber size and numbers within muscle bundle during the embryonic and post-hatch ages.

Impacts
What was accomplished under these goals? In avian species, positive relationships between egg weight (EW) and body weight (BW) have been reported. However, the correlation between the body growth rate and different weights of eggs from genetically mutated avian species was not studied yet. Myostatin (Mstn), an anti-myogenic factor, mutant quail were recently developed, and it was reported that EW produced from Mstn mutant quail (HO) was heavier compared to those from wild-type quail (WT). In the current study, distributions of pre-incubated EW and associations between EW and BW were compared between the Mstn mutant and WT quail lines. Average egg weight for the HO group was significantly heavier than the WT (p < 0.001) and the number of eggs having heavier EW (over 11g) were higher in the HO compared to the WT (p < 0.01). BW at week (W) 0, 4, and 6 after hatch were also significantly greater in the HO (p < 0.001 in all groups). In addition, linear regression analyses revealed positive relationships between EW and BW from W0 to W6, regardless of sex and genotype. Furthermore, Mstn mutant quail have heavier BW compared to the WT quail originated from eggs with similar weights. These data indicate that increased BW by Mstn mutation is contributed by increased EW and/or growth promoting activity of Mstn mutation independent of increasing egg sizes. These findings provide Mstn as a desirable genetic factor for selection of poultry breeds with superior growth. In addition, the knowledge gained from this study can inspire similar proof-of-concept studies involving standard and commercial lines of poultry. Recently developed myostatin (MSTN) mutant quail demonstrated similar effects of MSTN on muscle and fat developments between avian and mammalian species. However, the effect of MSTN mutation on the quality of eggshells, an important avian specific characteristic, has not yet been investigated although egg production traits of mutant quail have been studied. In this study, several parameters for eggshell quality, including eggshell size, eggshell weight, eggshell breaking strength (EBS), and eggshell thickness, were all compared between MSTN mutant and wild-type (WT) eggs. MSTN mutant eggs had greater height and width along with heavier eggshell weight compared to WT eggs, which shows a proportional improvement in egg size as affected by the MSTN mutation. However, EBS and eggshell thickness were decreased in mutant eggs compared to WT eggs. In addition, the palisade layer, the thickest and most important layer for the strength of an eggshell, was also decreased without a change in the number of vesicular holes. These data indicated that decreases in the thickness of the eggshell and the palisade layer would be a main factor contributing to a lower EBS in mutant eggs. MSTN mutant quail provide a useful model to better understand the function of MSTN on avian uterine cell development and eggshell biomineralization.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Lee J, McCurdy C, Chae C, Hwang J, Karolak MC, Kim DH, Baird CL, Bohrer BM, Lee K. 2021. Myostatin Mutation in Japanese Quail Increased Egg Size but Reduced Eggshell Thickness and Strength. Animals. 12(1):47.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Kim DH, Lee J, Choi YM, Lee K. 2022. Growth Promoting Potential in Mstn Mutant Quail Independent or Dependent of Increased Egg Size. Poultry Science.

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, biotechnology, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the food animal industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 1 postdoc, 1 technician, and 1 undergraduate student were trained to learn concepts of genome editing and assay skills and tasks for phenotyping. The genome editing concept they learned included embryo handing, micro injection, development of embryos into adult birds, and breeding of chimeras. The assay skills include poultry handling, tissue sampling, quantitative real-time PCR, Western blot, and analysis of histology of muscle tissues. The project provided opportunities to the researchers to learn muscle biology, developmental biology, molecular biology, and reproduction by investigating effects of MSTN mutation on physiological changes in quail. By understanding the physiological differences by genetic mutation, researchers also grasp the importance of integration of multiple research areas in advancing our knowledge in improving the poultry industry. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the project were published in scientific journals to be available to the scientific community in animal and poultry sciences. The results from the project were also presented in the virtual scientific meetings at the Ohio Agricultural Research and Development Center and local meetings. What do you plan to do during the next reporting period to accomplish the goals?For the last part of the project, different size of eggs from MSTN+/+, +/-, and -/- groups will be collected to compare body and muscle weight and fiber size and numbers.

Impacts
What was accomplished under these goals? Myostatin (MSTN) negatively regulates in muscle growth and development. This anti-myogenic function of MSTN has been revealed by increased muscle mass of mammals and fishes having MSTN mutations. For this project, MSTN mutation was generated using the CRISPR/Cas9 system in quail to investigate MSTN function in avian species. Body weight of MSTN homozygous mutant (MSTN-/-) quail became significantly heavier from 2 weeks old in males and 3 weeks old compared to MSTN heterozygous mutant (MSTN+/-) and wild-type (MSTN+/+) quail. In addition, MSTN-/- showed significantly heavier muscle mass compared to MSTN+/- and MSTN+/+. Especially, increased total fiber number of pectoralis major and gastrocnemius muscles without change in the muscle fiber cross-sectional area showed that muscle fiber hyperplasia in skeletal muscle resulted in increased body weight and muscle mass of MSTN-/- quail. To investigate feed efficiency, food conversion ration (FCR) was calculated by measuring body weight gain (WG) and feed intake (FI) from post-hatching day (D) 10 to D20, from D20 to D30 and from D30 to D40. FCR from D30 to D40 and overall period (from D10 to D40) were significantly lower in MSTN-/- compared to MSTN +/+ indicating improved feed efficiency by MSTN mutation in both the male and female. In terms of egg laying characteristics, MSTN-/- laid significantly heavier eggs compared to MSTN+/+. However, onset of egg laying was delayed, and eggs numbers were reduced. These data about MSTN function in growth performance and egg production of quail provide a scientific basis for potential usage of MSTN expression as a selection marker in broiler and layer industries.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lee JB, Kim DH, Lee K. 2020. Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide. International Journal of Molecular Science. 21:E1504.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lee J, Kim D-H, Brower AM, Schlachter I, Lee K. 2021. Research Note: Improved feed efficiency in quail with targeted genome editing in the myostatin gene. Poultry Science. 100(8):101257.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lee J, Kim D-H, Brower AM, Schlachter I, Lee K. 2021. Effects of Myostatin Mutation on Onset of Laying, Egg Production, Fertility, and Hatchability. Animals. 11(7): 1935.