Objective Since the first discovery that CRISPR-Cas system provides adaptive immunity of prokaryotic hosts to virus and other mobile genetic elements (MGEs). Numerous studies yielded vital insights into the immune mechanisms, and CRISPR-Cas system has been wildly utilized in gene editing and related research efforts. In the three major immune stages—adaptation, expression and maturation, interference spacer sequences play important roles separately. Although PAM (protospacer adjacent motif) determines the identification of targeted genes by CRISPR-Cas system, what drives the selection of specific genes and reservation on the CRISPR array remains uncertain. To explore the targeting characteristics of CRISPR-Cas systems, the virome of Yangshan harbor surface water and the CRISPR-Cas spacers available in public datasets were subjected to analysis.Methods Based on BLAST searching, viral sequence identification, gene function prediction, and gene conserved domain annotation, the final analysis results were obtained.Results As a result, 25 391 double-stranded DNA viral sequences were identified in the virome; 265 open reading frames (ORFs) were predicted for 238 sequences, and 134 CRISPR-Cas spacer sequences yielded 315 viral hits. 128 viral ORFs and 135 hits were functionally annotated, and the top 5 hits including terminase, capsid protein, portal protein, peptidase, and DNA methyltransferase. The matching of spacer (host)-protospacer (virus) often occurred in conserved domains or key structural domains of viral functional genes. Meanwhile, the number of CRISPR-Cas spacer sequence matches for Type_I systems under the Class 1 was much higher than that for other types of systems, accounting for 89.0% of the total. The results show that the CRISPR system will specifically identify and act on key functional genes of the virus.Conclusion The results of this study reveal the targeting specificity of the CRISPR-Cas system, showing new insights and providing new evidence for a better understanding of the mechanisms of virus-host immune interactions.