[논문]Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect

Lee, Jeong Hyo; Kim, Si Won; Park, Tae Sub

doi:10.5713/ajas.16.0695

Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect 원문보기

Asian-Australasian journal of animal sciences, v.30 no.5, 2017년, pp.743 - 748

Lee, Jeong Hyo (Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University) , Kim, Si Won (Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University) , Park, Tae Sub (Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University)

Abstract ▼ AI-Helper

Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

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제안 방법

Thus, a better understanding of skeletal muscle growth and differentiation mechanism could be directly applied to agribio industry. In this study, we examined and verified the Cas9-D10A nickase-mediated genomic editing in chicken cells to investigate the functional activity and regulatory mechanism. In the future, production of specific genome-tailored knockout and knockin cell lines will make important contributions via their use as in vitro models and in biofunctional studies in bioscience.
To investigate the non-specific off-target effect produced by the chicken myostatin knockout gRNAs, the predicted off-target sites were amplified and sequenced. Based on the chicken genomic sequences (galGal4), the off-target sites were analyzed and predicted on the web site of http://crispr.

참고문헌 (24)

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Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect 원문보기

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Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect 원문보기

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