Determination of Transgenes in Crops by a Strategy Combining Cas9 Targeting with Amplification-Free Long-Read Nanopore Sequencing

Considering any transgene in crops generated through genetic engineering is a potential safety concern, reliable methods for detecting transgenes are needed. Although whole-genome sequencing (WGS) using next-generation sequencing technology is a promising method for comprehensively detecting transgenes, its utility depends on the production of large amounts of sequencing data. Transgene enrichment may address this issue. We herein propose a strategy that combines amplification-free long-read nanopore sequencing with the CRISPR/Cas9 system. Specifically, the transgene is selectively enriched and analyzed by ligating an adapter after a target element of the transgene is cleaved by Cas9, which is followed by nanopore sequencing. In this study, we targeted three elements of transgenes frequently used to develop genetically modified (GM) and genome-edited (GE) crops. Our data showed that this strategy can detect transgenes more efficiently than WGS. Therefore, it may be useful in food safety assessment for GM and GE crops.