• CRISPRshuttle cloning enables high-throughput transfer of DNA fragments between vectors without PCR amplification, overcoming a major bottleneck in functional genomics.
• The method successfully cloned 1397 human genes into UAS vectors, demonstrating scalability and efficiency for genome-wide library construction.
• By eliminating PCR, CRISPRshuttle reduces cost and time while avoiding errors associated with amplification of long or difficult sequences.
• This approach facilitates the construction of GAL4/UAS-based overexpression libraries, accelerating functional studies in model organisms.