2025-03-21 九州大学
<関連情報>
- https://www.kyushu-u.ac.jp/ja/researches/view/1233
- https://www.kyushu-u.ac.jp/f/60976/25_0321_01.pdf
- https://www.cell.com/cell-genomics/fulltext/S2666-979X(25)00067-9
Cas9ニッカーゼの戦略的ターゲティングがタンデム遺伝子アレイを拡大する Strategic targeting of Cas9 nickase expands tandem gene arrays
Hiroaki Takesue ∙ Satoshi Okada ∙ Goro Doi ∙ Yuki Sugiyama ∙ Emiko Kusumoto ∙ Takashi Ito
Cell Genomics Published:March 20, 2025
DOI:https://doi.org/10.1016/j.xgen.2025.100811
Graphical abstract
Highlights
- BITREx uses Cas9 nickase to expand tandem gene arrays by break-induced replication
- BITREx can generate megabase-sized gene arrays in budding yeast
- Splint DNA allows BITREx to form a tandem array de novo from a single-copy gene
- BITREx is applicable to mammalian cells
Summary
Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here, we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has just replicated the array, forming a single-ended double-strand break. This break is subsequently end-resected to become single stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching ∼1 Mb. Furthermore, appropriate splint DNAs allow BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.
