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Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos
Weikai Ge1,2,3,4, Xiaozhu Zhao1,5, Shixue Gou1,2,3
1China-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.
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.
Insights
Cas9 protein alone, without guide RNAs, damages pig embryo genomes, causing cell death and developmental issues. This highlights risks of Cas9 protein in germline genome editing and gene therapy.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- CRISPR-Cas9 is a powerful gene-editing tool.
- The effects of Cas9 protein alone on embryonic development are not fully understood.
Purpose of the Study:
- To investigate the impact of Cas9 protein without guide RNAs on pig preimplantation embryo development.
- To assess genome integrity, apoptosis, and transcriptomic changes induced by Cas9 protein in embryos.
Main Methods:
- Cas9 mRNA was injected into pig zygotes.
- Embryos were cultured to the blastocyst stage.
- Genome damage was assessed by measuring phosphorylated histone H2AX foci.
- Apoptosis and cell numbers were quantified.
- Single-blastocyst RNA sequencing was performed.
Main Results:
- Cas9 protein persisted in embryos until the blastocyst stage.
- Cas9 protein alone induced DNA damage (increased H2AX foci) in preimplantation embryos.
- This led to increased apoptosis and reduced blastocyst cell numbers.
- Transcriptomic analysis revealed suppressed developmental pathways (cell cycle, metabolism, communication) and activated cell death pathways (apoptosis, necroptosis).
Conclusions:
- Cas9 protein alone can impair preimplantation embryonic development by inducing genome damage and altering gene expression.
- These findings underscore the importance of considering Cas9 protein's off-target effects in CRISPR-Cas9 applications, particularly in germline genome editing and gene therapy.
- Careful evaluation of Cas9 protein's impact is crucial for safe and effective therapeutic strategies.
