Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We pr...

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Autores principales:	Anastasia Yunusova, Alexander Smirnov, Tatiana Shnaider, Varvara Lukyanchikova, Svetlana Afonnikova, Nariman Battulin
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	auxin-inducible degron (AID) system CRISPR/Cas9 condensin cohesin mouse ES cells HAP1 cells Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/326ad3447e7448588d75862d96e9e9d8
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spelling	oai:doaj.org-article:326ad3447e7448588d75862d96e9e9d82021-11-04T06:25:49ZEvaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells2296-889X10.3389/fmolb.2021.757394https://doaj.org/article/326ad3447e7448588d75862d96e9e9d82021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmolb.2021.757394/fullhttps://doaj.org/toc/2296-889XThe auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.Anastasia YunusovaAlexander SmirnovTatiana ShnaiderVarvara LukyanchikovaSvetlana AfonnikovaSvetlana AfonnikovaNariman BattulinNariman BattulinFrontiers Media S.A.articleauxin-inducible degron (AID) systemCRISPR/Cas9condensincohesinmouse ES cellsHAP1 cellsBiology (General)QH301-705.5ENFrontiers in Molecular Biosciences, Vol 8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	auxin-inducible degron (AID) system CRISPR/Cas9 condensin cohesin mouse ES cells HAP1 cells Biology (General) QH301-705.5
spellingShingle	auxin-inducible degron (AID) system CRISPR/Cas9 condensin cohesin mouse ES cells HAP1 cells Biology (General) QH301-705.5 Anastasia Yunusova Alexander Smirnov Tatiana Shnaider Varvara Lukyanchikova Svetlana Afonnikova Svetlana Afonnikova Nariman Battulin Nariman Battulin Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
description	The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.
format	article
author	Anastasia Yunusova Alexander Smirnov Tatiana Shnaider Varvara Lukyanchikova Svetlana Afonnikova Svetlana Afonnikova Nariman Battulin Nariman Battulin
author_facet	Anastasia Yunusova Alexander Smirnov Tatiana Shnaider Varvara Lukyanchikova Svetlana Afonnikova Svetlana Afonnikova Nariman Battulin Nariman Battulin
author_sort	Anastasia Yunusova
title	Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_short	Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_full	Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_fullStr	Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_full_unstemmed	Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_sort	evaluation of the ostir1 and atafb2 aid systems for genome architectural protein degradation in mammalian cells
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/326ad3447e7448588d75862d96e9e9d8
work_keys_str_mv	AT anastasiayunusova evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT alexandersmirnov evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT tatianashnaider evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT varvaralukyanchikova evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT svetlanaafonnikova evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT svetlanaafonnikova evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT narimanbattulin evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells AT narimanbattulin evaluationoftheostir1andatafb2aidsystemsforgenomearchitecturalproteindegradationinmammaliancells
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Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

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