Strategic enhancement of Cas9 nickase-driven gene amplification

Gene amplification plays a critical role in evolution and disease and is widely utilized to overexpress valuable gene products in biotechnology. To broaden these applications, we previously developed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx), a method utilizing Cas9 nickase (nCas9) to amplify genetic sequences by driving tandem array expansion through ectopic BIR. Since BITREx efficiency depends on the guide RNA (gRNA) recruiting nCas9 to the array's flanking regions, here we develop a plasmid-based reporter system in budding yeast for the rapid identification of high-performing gRNAs. Furthermore, we introduce BITREx 2.0, a dual-nicking strategy that targets both sides of the gene array. We demonstrate that BITREx 2.0 is effective for both natural and synthetic arrays, enhancing expansion efficiency by up to an order of magnitude compared to the original single-nicking format. These advancements significantly broaden the applicability and efficiency of nCas9-mediated gene amplification across diverse biological and biotechnological contexts.