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

Yuanming Wang; Kaiwen Ivy Liu; Mengying Mandy Liu; Kean Hean Ooi; Tram Anh Nguyen; Jiunn En Chee; Shun Xiang Danny Teo; Shan He; Jie Wen Douglas Tay; Seok Yee Teo; Kai Shin Liew; Xiao Yu Ge; Zhi Jian Ng; Hasmik Avagyan; Hao Liu; Zirong Yi; Keziah Chang; Eng Piew Louis Kok; Runjia Chen; Chun En Yau; Jun Wei Koh; Yue Wan; Meng How Tan

doi:10.1038/s41587-024-02430-w

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

Yuanming Wang, Kaiwen Ivy Liu, Mengying Mandy Liu, Kean Hean Ooi, Tram Anh Nguyen, Jiunn En Chee, Shun Xiang Danny Teo, Shan He, Jie Wen Douglas Tay, Seok Yee Teo, Kai Shin Liew, Xiao Yu Ge, Zhi Jian Ng, Hasmik Avagyan, Hao Liu, Zirong Yi, Keziah Chang, Eng Piew Louis Kok, Runjia Chen, Chun En YauJun Wei Koh, Yue Wan, Meng How Tan^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Journal	Nature Biotechnology
DOIs	https://doi.org/10.1038/s41587-024-02430-w
Publication status	Accepted/In press - 2024
Externally published	Yes

Bibliographical note

Publisher Copyright:
© The Author(s), under exclusive licence to Springer Nature America, Inc. 2024.

ASJC Scopus Subject Areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

Access to Document

10.1038/s41587-024-02430-w

Cite this

Wang, Y., Liu, K. I., Liu, M. M., Ooi, K. H., Nguyen, T. A., Chee, J. E., Teo, S. X. D., He, S., Tay, J. W. D., Teo, S. Y., Liew, K. S., Ge, X. Y., Ng, Z. J., Avagyan, H., Liu, H., Yi, Z., Chang, K., Kok, E. P. L., Chen, R., ... Tan, M. H. (Accepted/In press). A circularly permuted CasRx platform for efficient, site-specific RNA editing. Nature Biotechnology. https://doi.org/10.1038/s41587-024-02430-w

@article{ac44ca4cd4ed423bb88b115662fe5bb5,

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

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.",

author = "Yuanming Wang and Liu, {Kaiwen Ivy} and Liu, {Mengying Mandy} and Ooi, {Kean Hean} and Nguyen, {Tram Anh} and Chee, {Jiunn En} and Teo, {Shun Xiang Danny} and Shan He and Tay, {Jie Wen Douglas} and Teo, {Seok Yee} and Liew, {Kai Shin} and Ge, {Xiao Yu} and Ng, {Zhi Jian} and Hasmik Avagyan and Hao Liu and Zirong Yi and Keziah Chang and Kok, {Eng Piew Louis} and Runjia Chen and Yau, {Chun En} and Koh, {Jun Wei} and Yue Wan and Tan, {Meng How}",

note = "Publisher Copyright: {\textcopyright} The Author(s), under exclusive licence to Springer Nature America, Inc. 2024.",

year = "2024",

doi = "10.1038/s41587-024-02430-w",

language = "English",

journal = "Nature Biotechnology",

issn = "1087-0156",

publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - A circularly permuted CasRx platform for efficient, site-specific RNA editing

AU - Wang, Yuanming

AU - Liu, Kaiwen Ivy

AU - Liu, Mengying Mandy

AU - Ooi, Kean Hean

AU - Nguyen, Tram Anh

AU - Chee, Jiunn En

AU - Teo, Shun Xiang Danny

AU - He, Shan

AU - Tay, Jie Wen Douglas

AU - Teo, Seok Yee

AU - Liew, Kai Shin

AU - Ge, Xiao Yu

AU - Ng, Zhi Jian

AU - Avagyan, Hasmik

AU - Liu, Hao

AU - Yi, Zirong

AU - Chang, Keziah

AU - Kok, Eng Piew Louis

AU - Chen, Runjia

AU - Yau, Chun En

AU - Koh, Jun Wei

AU - Wan, Yue

AU - Tan, Meng How

PY - 2024

Y1 - 2024

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85206385578&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85206385578&partnerID=8YFLogxK

U2 - 10.1038/s41587-024-02430-w

DO - 10.1038/s41587-024-02430-w

M3 - Article

C2 - 39385008

AN - SCOPUS:85206385578

SN - 1087-0156

JO - Nature Biotechnology

JF - Nature Biotechnology

ER -

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

Abstract

Bibliographical note

ASJC Scopus Subject Areas

Access to Document

Other files and links

Fingerprint

Cite this