Type III CRISPR-Cas systems preferentially acquire spacers from early-expressed phage genes in a transcription-dependent manner.

CRISPR-Cas immunity depends on acquiring spacers that generate functional CRISPR RNA (crRNA) guides. For most RNA-targeting type III systems, this poses a paradox: spacers are captured from DNA but defense requires RNA interaction, thus the system must infer transcriptional relevance of DNA fragments. We compared spacer acquisition profiles for co-occurring type III (RNA-recognizing) and type II (DNA-recognizing) systems in Streptococcus thermophilus during lytic phage infection. Type III spacer acquisition was highly enriched for early phage genes whereas type II spacers were distributed genome wide. High-throughput spacer sequencing showed that the type III early gene bias arose during adaptation, was independent of interference, and peaked immediately downstream of a conserved early phage promoter. This bias for early phage promoters was also observed in natural type III spacers identified in publicly available S. thermophilus genomes. Cloning this early phage promoter and downstream gene into a plasmid recreated the type III-acquisition hotspot. Mutation to disrupt the promoter eliminated the spacer hotspot, while mutation to disrupt translation enhanced it. Thus, type III-A adaptation preferentially samples DNA associated with the earliest phage transcriptional program, enriching for spacers that generate crRNAs targeting early phage RNA.