Guide RNA categorization enables target site choice in Tn7-CRISPR-Cas transposons

CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways, one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Selective regulation of specialized guide RNAs allows long-term memory for access to chromosomal sites upon entry into a new host, while conventional CRISPR features maintain the ability to continually acquire guide RNAs to new plasmid and phage targets. Transposon-encoded guide RNAs are also privatized to be recognized only by the transposon-adapted system working with selective regulation to guard against toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals new avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.