Strategic targeting of Cas9 nickase induces large segmental duplications

• DOI: 10.1016/j.xgen.2024.100610
• Publication Date: 2024-07-24T14:50:29Z
• Authors: Yuki Sugiyama; Satoshi Okada; Yasukazu Daigaku; Emiko Kusumoto; Takashi Ito

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 downstream of a replication origin on opposite strands. This configuration directs the sister forks initiated from to break at nicks, generating pair one-ended double-strand breaks. If homologous sequences flank sites, then end resection converts them single-stranded DNAs that readily anneal drive duplication region bounded by sequences. induces segments as large ∼1 Mb with efficiencies exceeding 10% budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint allow duplicate/multiplicate even not We also provide evidence mammalian cells. Therefore, provides prototype method induce structural variations manipulating fork progression.