Cas9缺口酶的策略性靶向诱导大片段重复。

IF 11.1 Q1 CELL BIOLOGY Cell genomics Pub Date : 2024-08-14 Epub Date: 2024-07-24 DOI:10.1016/j.xgen.2024.100610
Yuki Sugiyama, Satoshi Okada, Yasukazu Daigaku, Emiko Kusumoto, Takashi Ito
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引用次数: 0

摘要

基因/片段重复在基因组进化和变异中起着至关重要的作用。在这里,我们引入了配对缺口诱导扩增(PNAmp)技术,用于实验诱导。PNAmp 策略性地将两个 Cas9 挑刺酶分别置于复制原点的上游和下游的相对链上。这种配置会引导从原点开始的姐妹复制叉在缺口处断裂,产生一对单端双链断裂。如果同源序列位于两个断裂点的两侧,那么末端切除就会将它们转化为单链 DNA,这些单链 DNA 很容易退火,从而驱动同源序列所包围区域的复制。在芽殖酵母(Saccharomyces cerevisiae)中,PNAmp 能诱导大至 1 Mb 的区段复制,效率超过 10%。此外,适当的拼接 DNA 甚至可以让 PNAmp 复制/增殖没有同源序列限制的片段。我们还提供了哺乳动物细胞中存在 PNAmp 的证据。因此,PNAmp 提供了一种通过操纵复制叉进程诱导结构变异的原型方法。
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Strategic targeting of Cas9 nickase induces large segmental duplications.

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.

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