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Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fix...

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Autores principales: Wang, Longxiang, Xiao, Yuan, Wei, Xiaowei, Pan, Jimin, Duanmu, Deqiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253261/
https://www.ncbi.nlm.nih.gov/pubmed/34220774
http://dx.doi.org/10.3389/fmicb.2021.686008
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author Wang, Longxiang
Xiao, Yuan
Wei, Xiaowei
Pan, Jimin
Duanmu, Deqiang
author_facet Wang, Longxiang
Xiao, Yuan
Wei, Xiaowei
Pan, Jimin
Duanmu, Deqiang
author_sort Wang, Longxiang
collection PubMed
description Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.
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spelling pubmed-82532612021-07-03 Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti Wang, Longxiang Xiao, Yuan Wei, Xiaowei Pan, Jimin Duanmu, Deqiang Front Microbiol Microbiology Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia. Frontiers Media S.A. 2021-06-18 /pmc/articles/PMC8253261/ /pubmed/34220774 http://dx.doi.org/10.3389/fmicb.2021.686008 Text en Copyright © 2021 Wang, Xiao, Wei, Pan and Duanmu. 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 Microbiology
Wang, Longxiang
Xiao, Yuan
Wei, Xiaowei
Pan, Jimin
Duanmu, Deqiang
Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti
title Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti
title_full Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti
title_fullStr Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti
title_full_unstemmed Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti
title_short Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti
title_sort highly efficient crispr-mediated base editing in sinorhizobium meliloti
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253261/
https://www.ncbi.nlm.nih.gov/pubmed/34220774
http://dx.doi.org/10.3389/fmicb.2021.686008
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