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Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding
CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessit...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Life Science Alliance LLC
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8761495/ https://www.ncbi.nlm.nih.gov/pubmed/35022246 http://dx.doi.org/10.26508/lsa.202101078 |
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author | Ergünay, Tunahan Ayhan, Özgecan Celen, Arda B Georgiadou, Panagiota Pekbilir, Emre Abaci, Yusuf T Yesildag, Duygu Rettel, Mandy Sobhiafshar, Ulduz Ogmen, Anna Emre, NC Tolga Sahin, Umut |
author_facet | Ergünay, Tunahan Ayhan, Özgecan Celen, Arda B Georgiadou, Panagiota Pekbilir, Emre Abaci, Yusuf T Yesildag, Duygu Rettel, Mandy Sobhiafshar, Ulduz Ogmen, Anna Emre, NC Tolga Sahin, Umut |
author_sort | Ergünay, Tunahan |
collection | PubMed |
description | CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme’s turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity. |
format | Online Article Text |
id | pubmed-8761495 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Life Science Alliance LLC |
record_format | MEDLINE/PubMed |
spelling | pubmed-87614952022-01-26 Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding Ergünay, Tunahan Ayhan, Özgecan Celen, Arda B Georgiadou, Panagiota Pekbilir, Emre Abaci, Yusuf T Yesildag, Duygu Rettel, Mandy Sobhiafshar, Ulduz Ogmen, Anna Emre, NC Tolga Sahin, Umut Life Sci Alliance Research Articles CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme’s turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity. Life Science Alliance LLC 2022-01-12 /pmc/articles/PMC8761495/ /pubmed/35022246 http://dx.doi.org/10.26508/lsa.202101078 Text en © 2022 Ergünay et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Articles Ergünay, Tunahan Ayhan, Özgecan Celen, Arda B Georgiadou, Panagiota Pekbilir, Emre Abaci, Yusuf T Yesildag, Duygu Rettel, Mandy Sobhiafshar, Ulduz Ogmen, Anna Emre, NC Tolga Sahin, Umut Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding |
title | Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding |
title_full | Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding |
title_fullStr | Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding |
title_full_unstemmed | Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding |
title_short | Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding |
title_sort | sumoylation of cas9 at lysine 848 regulates protein stability and dna binding |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8761495/ https://www.ncbi.nlm.nih.gov/pubmed/35022246 http://dx.doi.org/10.26508/lsa.202101078 |
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