Unveiling the Evolution of CRISPR Spacer Number: A Phylogenetic Analysis of its Correlation with Repeat Characteristics

Abstract

CRISPR-Cas systems in prokaryotes utilize spacers, segments of DNA acquired from invading phages, to guide immune defense mechanisms. This study investigates the evolution of CRISPR repertoire size by examining its relationships with repeat length, terminal repeat polymorphism, and structural stability in 1,958 bacterial genomes, identifying 5,465 CRISPR arrays. Using CRISPRCasFinder for annotation and RNAfold for predicting RNA secondary structures, we found significant variation in array characteristics. Long-repeat arrays ([≥]38 bp) showed a significant positive correlation between terminal repeat polymorphism and CRISPR spacer number, a correlation absent in short-repeat arrays (<38 bp), suggesting longer repeats facilitate recombination and spacer loss. Additionally, a negative correlation between repeat length and spacer number across all arrays indicates that longer repeats may accelerate spacer loss. Furthermore, our results show that immune demand significantly influences the evolution of spacer number. Larger CRISPR repertoires correlate with conserved repeat sequences and stable secondary structures, vital for functional arrays under continuous selective pressure. Comparing functional and obsolete CRISPR arrays (orphan arrays in genomes lacking Cas genes) revealed that obsolete arrays have fewer spacers and lower repeat consistency, indicating a degenerative state. By elucidating the factors that shape CRISPR memory size evolution, this research offers strategies to enhance bacterial defenses, mitigate resistance, and improve applications in gene editing and therapeutics.