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Cas9 核酸酶的靶向性诱导大片段重复。

Strategic targeting of Cas9 nickase induces large segmental duplications.

机构信息

Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan.

Cancer Genome Dynamics Project, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.

出版信息

Cell Genom. 2024 Aug 14;4(8):100610. doi: 10.1016/j.xgen.2024.100610. Epub 2024 Jul 24.

DOI:10.1016/j.xgen.2024.100610
PMID:39053455
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11406185/
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.

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，我们介绍了配对缺口诱导扩增（PNAmp）技术，用于它们的实验诱导。PNAmp 策略性地在复制起点的两条互补链的上下游位置放置两个 Cas9 切口酶。这种构象引导从起点起始的姐妹复制叉在缺口处断裂，产生一对单链双链断裂。如果同源序列侧翼位于两个断裂位点，则末端切除将它们转化为单链 DNA，这些单链 DNA 易于退火，从而驱动同源序列所限定区域的复制。PNAmp 可诱导大小达 1Mb 左右的片段重复，在酿酒酵母（Saccharomyces cerevisiae）中的效率超过 10%。此外，适当的衔接 DNA 允许 PNAmp 复制/倍增即使没有同源序列限制的片段。我们还提供了 PNAmp 在哺乳动物细胞中的证据。因此，PNAmp 通过操纵复制叉的进展提供了一种诱导结构变异的原型方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/4945b7f4eb8b/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/85b477b21b27/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/0e7f3e33ba4f/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/3d5a27ba1032/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/4da9e5e3a734/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/bae08f545a29/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/efbc97c8b953/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/818e7aaa5c6c/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/4945b7f4eb8b/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/85b477b21b27/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/0e7f3e33ba4f/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/3d5a27ba1032/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/4da9e5e3a734/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/bae08f545a29/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/efbc97c8b953/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/818e7aaa5c6c/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4d62/11406185/4945b7f4eb8b/gr7.jpg

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