Bacteriophages (phages) have emerged as promising tools for a variety of applications, including antibacterial agents, biomaterials, and vaccine development. However, wild-type phages are often unsuitable for these purposes. Recently, we have developed the second generation of CRISPR-based phage genome engineering technology using the CRISPR-Cas12a system, which can rapidly generate phage variants with unique properties to enhance their applications. This technology consists of a CRISPR-Cas12a plasmid expressing the type V CRISPR-Cas complex and a donor plasmid containing a desired mutation. The CRISPR-Cas complex creates a DNA double-strand break (DSB) at the specific site on the phage genome determined by the guide RNA (gRNA). The DSB is then repaired by homologous recombination with the donor to generate mutants of interest. This second-generation technology can more efficiently engineer the phages whose genomic DNAs are heavily modified, such as T4 phage. We provide here a step-by-step protocol to delete 10.8 Kb in T4 genome using Cas12a.