title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks.
abstract: 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.
authors.affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam, 1105 AZ, The Netherlands Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Lianne E M
authors.initials: LE
authors.lastname: Vriend
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Rohit
authors.initials: R
authors.lastname: Prakash
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA Weill Cornell Graduate School of Medical Sciences, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Chun-Chin
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Fabio
authors.initials: F
authors.lastname: Vanoli
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Cavallo
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA Weill Cornell Graduate School of Medical Sciences, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA m-jasin@ski.mskcc.org.
authors.forename: Maria
authors.initials: M
authors.lastname: Jasin
authors.affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam, 1105 AZ, The Netherlands Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Przemek M
authors.initials: PM
authors.lastname: Krawczyk
mesh_terms: D000818:Animals; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D059766:DNA End-Joining Repair; D004261:DNA Replication; D004720:Endonucleases / Q000378:metabolism*; D055786:Gene Knockout Techniques; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D059372:Nucleotide Motifs; D059767:Recombinational DNA Repair; D012854:Sister Chromatid Exchange
chemical_list: D004720:Endonucleases