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Strategic targeting of Cas9 nickase induces large segmental duplications.
Sugiyama, Yuki; Okada, Satoshi; Daigaku, Yasukazu; Kusumoto, Emiko; Ito, Takashi.
Affiliation
  • Sugiyama Y; Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan.
  • Okada S; Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan.
  • Daigaku Y; Cancer Genome Dynamics Project, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
  • Kusumoto E; Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan.
  • Ito T; Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan. Electronic address: ito.takashi.352@m.kyushu-u.ac.jp.
Cell Genom ; 4(8): 100610, 2024 Aug 14.
Article de En | MEDLINE | ID: mdl-39053455
ABSTRACT
Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.
Sujet(s)

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Saccharomyces cerevisiae Limites: Humans Langue: En Journal: Cell Genom Année: 2024 Type de document: Article Pays d'affiliation: Japon Pays de publication: États-Unis d'Amérique

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Saccharomyces cerevisiae Limites: Humans Langue: En Journal: Cell Genom Année: 2024 Type de document: Article Pays d'affiliation: Japon Pays de publication: États-Unis d'Amérique