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Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided...

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Autores principales: Vriend, Lianne E.M., Prakash, Rohit, Chen, Chun-Chin, Vanoli, Fabio, Cavallo, Francesca, Zhang, Yu, Jasin, Maria, Krawczyk, Przemek M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914091/
https://www.ncbi.nlm.nih.gov/pubmed/27001513
http://dx.doi.org/10.1093/nar/gkw179
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author Vriend, Lianne E.M.
Prakash, Rohit
Chen, Chun-Chin
Vanoli, Fabio
Cavallo, Francesca
Zhang, Yu
Jasin, Maria
Krawczyk, Przemek M.
author_facet Vriend, Lianne E.M.
Prakash, Rohit
Chen, Chun-Chin
Vanoli, Fabio
Cavallo, Francesca
Zhang, Yu
Jasin, Maria
Krawczyk, Przemek M.
author_sort Vriend, Lianne E.M.
collection PubMed
description DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing.
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spelling pubmed-49140912016-06-22 Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks Vriend, Lianne E.M. Prakash, Rohit Chen, Chun-Chin Vanoli, Fabio Cavallo, Francesca Zhang, Yu Jasin, Maria Krawczyk, Przemek M. Nucleic Acids Res Genome Integrity, Repair and Replication DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing. Oxford University Press 2016-06-20 2016-03-21 /pmc/articles/PMC4914091/ /pubmed/27001513 http://dx.doi.org/10.1093/nar/gkw179 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Genome Integrity, Repair and Replication
Vriend, Lianne E.M.
Prakash, Rohit
Chen, Chun-Chin
Vanoli, Fabio
Cavallo, Francesca
Zhang, Yu
Jasin, Maria
Krawczyk, Przemek M.
Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
title Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
title_full Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
title_fullStr Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
title_full_unstemmed Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
title_short Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
title_sort distinct genetic control of homologous recombination repair of cas9-induced double-strand breaks, nicks and paired nicks
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914091/
https://www.ncbi.nlm.nih.gov/pubmed/27001513
http://dx.doi.org/10.1093/nar/gkw179
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