Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

Weikai Ge; Xiaozhu Zhao; Shixue Gou; Qin Jin; Fangbing Chen; Zhen Ouyang; Chengdan Lai; Tao Cui; Baoyi Mai; Sijia Lu; Kexin Zhong; Yanhui Liang; Tao Chen; Han Wu; Nan Li; Yinghua Ye; Liangxue Lai; Kepin Wang

doi:10.1016/j.omtn.2023.102035

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

Mol Ther Nucleic Acids. 2023 Sep 20:34:102035. doi: 10.1016/j.omtn.2023.102035. eCollection 2023 Dec 12.

Authors

Weikai Ge^{1

2

3

4}, Xiaozhu Zhao^{1

5}, Shixue Gou^{1

2

3}, Qin Jin^{1

2

3}, Fangbing Chen^{1

2

3

4}, Zhen Ouyang^{1

2

3

4}, Chengdan Lai^{1

2

3

4}, Tao Cui⁶, Baoyi Mai³, Sijia Lu³, Kexin Zhong³, Yanhui Liang^{1

2

3}, Tao Chen⁴, Han Wu^{1

2

3}, Nan Li^{1

2

3}, Yinghua Ye^{1

2

3}, Liangxue Lai^{1

2

3

4}, Kepin Wang^{1

2

3

4}

Affiliations

¹ China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
² Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou 510530, China.
³ Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China.
⁴ Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529020, China.
⁵ University of Chinese Academy of Sciences, Beijing 100049, China.
⁶ College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.

Abstract

Cas9 protein without sgRNAs can induce genomic damage at the cellular level in vitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

Keywords: MT: RNA/DNA Editing; genomic damage; germline genome editing; guide-free Cas9; pig embryo; transcriptome changes.