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.

• PMID: 40118067
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Journal Article

Authors

Takesue H, Okada S, Doi G, Sugiyama Y, Kusumoto E, Ito T

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