The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

Abstract CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. I...

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Autores principales:	Minkyung Choi, Jae-Young Yun, Jun-Hyuk Kim, Jin-Soo Kim, Sang-Tae Kim
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9ab4f0e0f49c4448a11d44ca1dda3449
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spelling	oai:doaj.org-article:9ab4f0e0f49c4448a11d44ca1dda34492021-12-02T14:26:20ZThe efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana10.1038/s41598-021-87669-y2045-2322https://doaj.org/article/9ab4f0e0f49c4448a11d44ca1dda34492021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87669-yhttps://doaj.org/toc/2045-2322Abstract CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.Minkyung ChoiJae-Young YunJun-Hyuk KimJin-Soo KimSang-Tae KimNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Minkyung Choi Jae-Young Yun Jun-Hyuk Kim Jin-Soo Kim Sang-Tae Kim The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana
description	Abstract CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.
format	article
author	Minkyung Choi Jae-Young Yun Jun-Hyuk Kim Jin-Soo Kim Sang-Tae Kim
author_facet	Minkyung Choi Jae-Young Yun Jun-Hyuk Kim Jin-Soo Kim Sang-Tae Kim
author_sort	Minkyung Choi
title	The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana
title_short	The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana
title_full	The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana
title_fullStr	The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana
title_full_unstemmed	The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana
title_sort	efficacy of crispr-mediated cytosine base editing with the rps5a promoter in arabidopsis thaliana
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/9ab4f0e0f49c4448a11d44ca1dda3449
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The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

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