Conjugative plasmids can be used to deliver CRISPR systems into bacterial cells and interrogate their genomes for the presence of specific sequences. Target DNA sequences can be cleaved to cause cell death, hence enabling the use of CRISPR to fight infections or edit the microbiome. High transfer rates are critical to ensure the CRISPR system is delivered into as many bacterial cells as possible. We present a protocol for significantly increasing the transfer rates of a conjugative plasmid to desired bacterial strains or species. This is accomplished by accelerated laboratory evolution, which involves a mutagenic helper plasmid and successive cycles of bacterial conjugation as a selective pressure to isolate high-efficiency conjugative plasmid variants. The method is showcased by increasing the transfer rate of conjugative plasmid TP114 from E. coli Nissle 1917 to E. coli LF82, a model strain for inflammatory bowel diseases. This protocol can easily be adapted for other conjugative elements and bacterial species.