A circularly permuted CasRx platform for efficient, site-specific RNA editing

doi:10.1038/s41587-024-02430-w

. 2024 Oct 9.

doi: 10.1038/s41587-024-02430-w. Online ahead of print.

A circularly permuted CasRx platform for efficient, site-specific RNA editing

Yuanming Wang^#^{1

2}, Kaiwen Ivy Liu^#^{1

2}, Mengying Mandy Liu^#^{1

2}, Kean Hean Ooi^#^{1

2}, Tram Anh Nguyen¹, Jiunn En Chee^{1

2

3}, Shun Xiang Danny Teo^{1

2

4}, Shan He^{2

3}, Jie Wen Douglas Tay^{1

3}, Seok Yee Teo^{1

3}, Kai Shin Liew¹, Xiao Yu Ge¹, Zhi Jian Ng¹, Hasmik Avagyan¹, Hao Liu¹, Zirong Yi¹, Keziah Chang², Eng Piew Louis Kok², Runjia Chen^{2

5}, Chun En Yau^{2

6}, Jun Wei Koh^{2

6}, Yue Wan^{2

3

7}, Meng How Tan^{8

9

10}

Affiliations

¹ School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore.
² Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore.
³ School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
⁴ HP-NTU Digital Manufacturing Corporate Lab, Nanyang Technological University, Singapore, Singapore.
⁵ National Junior College, Singapore, Singapore.
⁶ Hwa Chong Institution, Singapore, Singapore.
⁷ Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
⁸ School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore. mh.tan@ntu.edu.sg.
⁹ Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore. mh.tan@ntu.edu.sg.
¹⁰ HP-NTU Digital Manufacturing Corporate Lab, Nanyang Technological University, Singapore, Singapore. mh.tan@ntu.edu.sg.

^# Contributed equally.

PMID: 39385008
DOI: 10.1038/s41587-024-02430-w

A circularly permuted CasRx platform for efficient, site-specific RNA editing

Yuanming Wang et al. Nat Biotechnol. 2024.

. 2024 Oct 9.

doi: 10.1038/s41587-024-02430-w. Online ahead of print.

Authors

Affiliations

¹ School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore.
² Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore.
³ School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
⁴ HP-NTU Digital Manufacturing Corporate Lab, Nanyang Technological University, Singapore, Singapore.
⁵ National Junior College, Singapore, Singapore.
⁶ Hwa Chong Institution, Singapore, Singapore.
⁷ Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
⁸ School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore. mh.tan@ntu.edu.sg.
⁹ Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore. mh.tan@ntu.edu.sg.
¹⁰ HP-NTU Digital Manufacturing Corporate Lab, Nanyang Technological University, Singapore, Singapore. mh.tan@ntu.edu.sg.

^# Contributed equally.

PMID: 39385008
DOI: 10.1038/s41587-024-02430-w

Abstract

Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function.

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Encoding spatial tumour dynamics with Starfysh.
He S. He S. Nat Rev Cancer. 2024 Oct 11. doi: 10.1038/s41568-024-00764-w. Online ahead of print. Nat Rev Cancer. 2024. PMID: 39394485 No abstract available.

References

1. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015). - PubMed - DOI
1. Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018). - PubMed - DOI
1. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018). - PubMed - DOI
1. Nishikura, K. A-to-I editing of coding and non-coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 17, 83–96 (2016). - PubMed - DOI
1. Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133–138 (2019). - PubMed - DOI

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[1] Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015). - PubMed - DOI

[2] Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015). - PubMed - DOI

[3] Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018). - PubMed - DOI

[4] Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018). - PubMed - DOI

[5] Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018). - PubMed - DOI

[6] Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018). - PubMed - DOI

[7] Nishikura, K. A-to-I editing of coding and non-coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 17, 83–96 (2016). - PubMed - DOI

[8] Nishikura, K. A-to-I editing of coding and non-coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 17, 83–96 (2016). - PubMed - DOI

[9] Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133–138 (2019). - PubMed - DOI

[10] Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133–138 (2019). - PubMed - DOI

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A circularly permuted CasRx platform for efficient, site-specific RNA editing

Affiliations

A circularly permuted CasRx platform for efficient, site-specific RNA editing

Authors

Affiliations

Abstract

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References

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Research Materials