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Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications o...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8921874/ https://www.ncbi.nlm.nih.gov/pubmed/35300420 http://dx.doi.org/10.3389/fcell.2022.809922 |
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author | Wu, Susu Li, Liping Li, Min Sun, Shiyu Zhao, Yuting Xue, Xiaowen Chen, Feiyu Zhong, Jingli Guo, Junfan Qu, Qianhui Wang, Xiongjun Liu, Zhen Qiao, Yunbo |
author_facet | Wu, Susu Li, Liping Li, Min Sun, Shiyu Zhao, Yuting Xue, Xiaowen Chen, Feiyu Zhong, Jingli Guo, Junfan Qu, Qianhui Wang, Xiongjun Liu, Zhen Qiao, Yunbo |
author_sort | Wu, Susu |
collection | PubMed |
description | CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively. |
format | Online Article Text |
id | pubmed-8921874 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-89218742022-03-16 Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo Wu, Susu Li, Liping Li, Min Sun, Shiyu Zhao, Yuting Xue, Xiaowen Chen, Feiyu Zhong, Jingli Guo, Junfan Qu, Qianhui Wang, Xiongjun Liu, Zhen Qiao, Yunbo Front Cell Dev Biol Cell and Developmental Biology CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively. Frontiers Media S.A. 2022-03-01 /pmc/articles/PMC8921874/ /pubmed/35300420 http://dx.doi.org/10.3389/fcell.2022.809922 Text en Copyright © 2022 Wu, Li, Li, Sun, Zhao, Xue, Chen, Zhong, Guo, Qu, Wang, Liu and Qiao. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Wu, Susu Li, Liping Li, Min Sun, Shiyu Zhao, Yuting Xue, Xiaowen Chen, Feiyu Zhong, Jingli Guo, Junfan Qu, Qianhui Wang, Xiongjun Liu, Zhen Qiao, Yunbo Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo |
title | Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
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title_full | Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
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title_fullStr | Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
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title_full_unstemmed | Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
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title_short | Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
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title_sort | two compact cas9 ortholog-based cytosine base editors expand the dna targeting scope and applications in vitro and in vivo |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8921874/ https://www.ncbi.nlm.nih.gov/pubmed/35300420 http://dx.doi.org/10.3389/fcell.2022.809922 |
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