Strategic targeting of Cas9 nickase expands tandem gene arrays

Takesue, Hiroaki, Okada, Satoshi, Doi, Goro, Sugiyama, Yuki, Kusumoto, Emiko, Ito, Takashi

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.

Strategic targeting of Cas9 nickase expands tandem gene arrays

Search this article

Description

Journal

Related Projects

Keywords

Details 詳細情報について

Export

Report a problem