The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.