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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
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.
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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
title_full Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
title_fullStr Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
title_full_unstemmed Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
title_short Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo
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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