Small CRISPR RNAs guide antiviral defense in prokaryotes. Prokaryotes acquire virus resistance by integrating short viral DNA fragments into CRISPR arrays. This study shows how CRISPR-associated (Cas) proteins use these viral sequences to mediate an antiviral response. After transcription of the CRISPR, a complex of Cas proteins called Cascade cleaves a CRISPR RNA precursor in each repeat, retaining the cleavage products containing the viral sequence. These mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. The study demonstrates that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense. The CRISPR system protects bacteria and archaea against viruses by integrating viral DNA sequences into CRISPR loci. These sequences function as a genetic memory to prevent infection by viruses containing the recognition sequence. The study identifies a complex of five Cas proteins, Cascade, responsible for the maturation of pre-crRNA to small crRNAs critical for antiviral response. These mature crRNAs contain the antiviral spacer unit flanked by short RNA sequences derived from the repeat on either side, termed the 5' and 3' handles. The Cascade-bound crRNA serves as a guide to direct the complex to viral nucleic acids to mediate an antiviral response. The study shows that pre-crRNA cleavage is mechanistically required for phage resistance, and that both Cascade and Cas3 are required in this process. The results demonstrate that the transcription of CRISPR regions and the cleavage of pre-crRNA to mature crRNAs by Cas proteins is the molecular basis of the antiviral defense stage of the CRISPR/cas system.Small CRISPR RNAs guide antiviral defense in prokaryotes. Prokaryotes acquire virus resistance by integrating short viral DNA fragments into CRISPR arrays. This study shows how CRISPR-associated (Cas) proteins use these viral sequences to mediate an antiviral response. After transcription of the CRISPR, a complex of Cas proteins called Cascade cleaves a CRISPR RNA precursor in each repeat, retaining the cleavage products containing the viral sequence. These mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. The study demonstrates that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense. The CRISPR system protects bacteria and archaea against viruses by integrating viral DNA sequences into CRISPR loci. These sequences function as a genetic memory to prevent infection by viruses containing the recognition sequence. The study identifies a complex of five Cas proteins, Cascade, responsible for the maturation of pre-crRNA to small crRNAs critical for antiviral response. These mature crRNAs contain the antiviral spacer unit flanked by short RNA sequences derived from the repeat on either side, termed the 5' and 3' handles. The Cascade-bound crRNA serves as a guide to direct the complex to viral nucleic acids to mediate an antiviral response. The study shows that pre-crRNA cleavage is mechanistically required for phage resistance, and that both Cascade and Cas3 are required in this process. The results demonstrate that the transcription of CRISPR regions and the cleavage of pre-crRNA to mature crRNAs by Cas proteins is the molecular basis of the antiviral defense stage of the CRISPR/cas system.