Pooled CRISPR libraries are widely used in high-throughput screening to study various biological processes. Various pooled CRISPR libraries have been shared for CRISPR screens and useful tools have been developed to construct researcher's own libraries, however, many researchers are struggling to create their own pooled CRISPR libraries: it is a time-consuming, labor-intensive, and expensive process. In this study, we develop a simple method to customize conventional pooled CRISPR libraries using the CRISPR/Cas9 system. We show that conventional pooled CRISPR libraries can be modified by eliminating gRNAs that target positive genes, enabling the identification of unknown target genes in CRISPR screening. CRISPR/Cas9 system can be applied as a precise tool for customizing conventional pooled CRISPR libraries and will broaden the scope of high-throughput screening technology.