Dataset Information

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

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.

SUBMITTER: Ge W

PROVIDER: S-EPMC10551558 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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

Ge Weikai W Zhao Xiaozhu X Gou Shixue S Jin Qin Q Chen Fangbing F Ouyang Zhen Z Lai Chengdan C Cui Tao T Mai Baoyi B Lu Sijia S Zhong Kexin K Liang Yanhui Y Chen Tao T Wu Han H Li Nan N Ye Yinghua Y Lai Liangxue L Wang Kepin K

Molecular therapy. Nucleic acids 20230920

Cas9 protein without sgRNAs can induce genomic damage at the cellular level <i>in vitro</i>. 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 chromati ...[more]

PMID: 37808924

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Project description:DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.

Dataset Information

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

Publications

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

Similar Datasets

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