BITREx induced Yeast population
Expanding tandem gene arrays has played a critical role in creating multigene families and will likely contribute to constructing stable gene overexpression systems. We have developed BITREx, a novel method for expanding tandem gene arrays. By targeting Cas9 nickase to a tandem repeat-flanking site, BITREx collapses the replication fork that has just replicated the repeat to form a one-ended double-stranded break, from which end resection generates single-stranded DNA (ssDNA). Because there is no repeat unit downstream of the break, the ssDNA often invades upstream units to initiate break-induced replication that expands the tandem repeat. Indeed, BITREx successfully extended a ~32-kb CUP1 array consisting of 16 copies of a 2-kb repeat unit encoding a copper metallothionein Cup1 to a population average of ~1 Mb or ~500 repeat units after 30 days of culture without selection pressure of copper, suggesting its potential utility in gene overexpression.