A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development

doi:10.1007/s11427-023-2572-6

. 2024 Nov;67(11):2297-2306.

doi: 10.1007/s11427-023-2572-6. Epub 2024 Aug 2.

A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development

Lin Zhang^#¹, Shi-Meng Cao^#², Hao Wu², Meng Yan¹, Jinsong Li^{3

4

5}, Ling-Ling Chen^{6

7

8

9}

Affiliations

¹ State 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.
² Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
³ State 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. jsli@sibcb.ac.cn.
⁴ Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China. jsli@sibcb.ac.cn.
⁵ School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. jsli@sibcb.ac.cn.
⁶ Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. linglingchen@sibcb.ac.cn.
⁷ Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China. linglingchen@sibcb.ac.cn.
⁸ School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. linglingchen@sibcb.ac.cn.
⁹ New Cornerstone Science Laboratory, Shenzhen, 518054, China. linglingchen@sibcb.ac.cn.

^# Contributed equally.

PMID: 39110403
DOI: 10.1007/s11427-023-2572-6

A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development

Lin Zhang et al. Sci China Life Sci. 2024 Nov.

. 2024 Nov;67(11):2297-2306.

doi: 10.1007/s11427-023-2572-6. Epub 2024 Aug 2.

Authors

Lin Zhang^#¹, Shi-Meng Cao^#², Hao Wu², Meng Yan¹, Jinsong Li^{3

4

5}, Ling-Ling Chen^{6

7

8

9}

Affiliations

¹ State 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.
² Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
³ State 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. jsli@sibcb.ac.cn.
⁴ Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China. jsli@sibcb.ac.cn.
⁵ School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. jsli@sibcb.ac.cn.
⁶ Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. linglingchen@sibcb.ac.cn.
⁷ Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China. linglingchen@sibcb.ac.cn.
⁸ School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. linglingchen@sibcb.ac.cn.
⁹ New Cornerstone Science Laboratory, Shenzhen, 518054, China. linglingchen@sibcb.ac.cn.

^# Contributed equally.

PMID: 39110403
DOI: 10.1007/s11427-023-2572-6

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.

Keywords: CRISPR-RfxCas13d; RNA knockdown; embryonic development; semi-cloned technology; sperm-like stem cell.

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References

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[1] Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D. B.T., Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573. - DOI - PubMed - PMC

[2] Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D. B.T., Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573. - DOI - PubMed - PMC

[3] Bai, M., Han, Y., Wu, Y., Liao, J., Li, L., Wang, L., Li, Q., Xing, W., Chen, L., Zou, W., et al. (2019). Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development. PLoS Biol 17, e3000350. - DOI - PubMed - PMC

[4] Bai, M., Han, Y., Wu, Y., Liao, J., Li, L., Wang, L., Li, Q., Xing, W., Chen, L., Zou, W., et al. (2019). Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development. PLoS Biol 17, e3000350. - DOI - PubMed - PMC

[5] Batra, R., Nelles, D.A., Pirie, E., Blue, S.M., Marina, R.J., Wang, H., Chaim, I.A., Thomas, J.D., Zhang, N., Nguyen, V., et al. (2017). Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell 170, 899–912.e10. - DOI - PubMed - PMC

[6] Batra, R., Nelles, D.A., Pirie, E., Blue, S.M., Marina, R.J., Wang, H., Chaim, I.A., Thomas, J.D., Zhang, N., Nguyen, V., et al. (2017). Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell 170, 899–912.e10. - DOI - PubMed - PMC

[7] East-Seletsky, A., O’Connell, M.R., Knight, S.C., Burstein, D., Cate, J.H.D., Tjian, R., and Doudna, J.A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273. - DOI - PubMed - PMC

[8] East-Seletsky, A., O’Connell, M.R., Knight, S.C., Burstein, D., Cate, J.H.D., Tjian, R., and Doudna, J.A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273. - DOI - PubMed - PMC

[9] Fire, A., Xu, S.Q., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. - DOI - PubMed

[10] Fire, A., Xu, S.Q., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. - DOI - PubMed

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A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development

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A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development

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