Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells
© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc..
Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity-CDG-nCas9 and TDG-nCas9-with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5' end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2024 |
|---|---|
| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2024 |
|---|---|
| Enthalten in: |
Nature biotechnology - (2024) vom: 02. Jan. |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Ye, Lijun [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
|---|
| Anmerkungen: |
Date Revised 03.01.2024 published: Print-Electronic Citation Status Publisher |
|---|
| doi: |
10.1038/s41587-023-02050-w |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM366596195 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM366596195 | ||
| 003 | DE-627 | ||
| 005 | 20240108141843.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 240108s2024 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1038/s41587-023-02050-w |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1248.xml |
| 035 | |a (DE-627)NLM366596195 | ||
| 035 | |a (NLM)38168994 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Ye, Lijun |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells |
| 264 | 1 | |c 2024 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Revised 03.01.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status Publisher | ||
| 520 | |a © 2024. The Author(s), under exclusive licence to Springer Nature America, Inc. | ||
| 520 | |a Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity-CDG-nCas9 and TDG-nCas9-with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5' end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects | ||
| 650 | 4 | |a Journal Article | |
| 700 | 1 | |a Zhao, Dongdong |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Ju |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Yiran |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Bo |e verfasserin |4 aut | |
| 700 | 1 | |a Yang, Yuanzhao |e verfasserin |4 aut | |
| 700 | 1 | |a Hou, Xueting |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Huibin |e verfasserin |4 aut | |
| 700 | 1 | |a Wei, Zhandong |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Xiaoqi |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Yaqiu |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Siwei |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Yajing |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Xueli |e verfasserin |4 aut | |
| 700 | 1 | |a Bi, Changhao |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Nature biotechnology |d 1996 |g (2024) vom: 02. Jan. |w (DE-627)NLM08998398X |x 1546-1696 |7 nnns |
| 773 | 1 | 8 | |g year:2024 |g day:02 |g month:01 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1038/s41587-023-02050-w |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |j 2024 |b 02 |c 01 | ||