CRISPR-Cas interference decays rapidly with distance from the leader sequence in a long array

Spacer efficacy generally declines with distance from the leader sequence, but the scarcity of fine-scale studies hampers comparisons across taxa. Here, we investigated positional effects across an exceptionally long 121-spacer CRISPR array associated with the type I-C cas operon of a Myxococcus xanthus natural isolate. In plasmid-interference assays, we found that interference rapidly declined with distance from the leader sequence, with only the proximal [~]4% of spacers conferring measurable interference. This contrasts strikingly with a study in Vibrio cholerae, in which it was shown that [~]95% of spacers in a shorter (39-spacer) array were effective. Our results suggest that there is great variation in the effective proportion of spacers across species, highlighting the need for fine-scale studies of CRISPR-array activity across diverse bacterial lineages.