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Updated: Jun 17, 2025
Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes
Published on: December 16, 2022
A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development
Lin Zhang1, Shi-Meng Cao2, Hao Wu2
1State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China.
Abstract:
The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.
Insights
This study introduces a novel method combining CRISPR-RfxCas13d and semi-cloning to suppress specific RNA levels in vivo. This technique effectively revealed critical roles of Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2 in embryonic development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Diverse RNA classes (mRNA, lncRNA, circRNA) are crucial for development and disease.
- Limited understanding of RNA functions in vivo due to lack of effective RNA manipulation tools.
Purpose of the Study:
- To develop and validate a novel system for targeted RNA suppression in live organisms.
- To investigate the roles of specific RNAs (Sfmbt2 mRNA, Fendrr lncRNA, circMan1a2) in embryonic development.
Main Methods:
- Utilized the CRISPR-RfxCas13d system for RNA targeting.
- Employed sperm-like stem cell-mediated semi-cloning for gene delivery and expression suppression.
- Interfered with the expression of Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2.
Main Results:
- Successfully suppressed the expression of targeted mRNA, lncRNA, and circRNA molecules.
- Demonstrated critical roles of these RNAs in embryonic development, evidenced by phenotypes like lethality and developmental delay.
- Observed embryo resorption upon suppression of specific RNA targets.
Conclusions:
- The developed CRISPR-RfxCas13d and semi-cloning method provides a powerful tool for targeted RNA silencing in vivo.
- This methodology enables functional studies of specific RNAs without genetic modification.
- The study highlights the essential roles of Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2 in successful embryonic development.
