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

Abstract

<jats:title>Summary</jats:title><jats:p>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.</jats:p>

Tn7-CRISPR-Cas12K elements manage pathway choice using truncated repeat-spacer units to target tRNA attachment sites

“…Our identification of distinct, att -targeting guide RNAs in Cas12K elements is similar to our previous finding with I-F3 elements 7 . Similar to the I-F3 elements, Cas12K elements possess att -targeting spacers, and the locations of att -targeting spacers are either at the end of CRISPR array or further downstream (Figure 2).…”

“…The I-F3 elements that show guide RNA categorization, also have a sister group that uses two TniQ proteins to allow the use of dual pathways; one that is dedicated to a parE chromosomal attachment site recognized by TniQ and the second functioning as a dedicated part of the I-F3 CRISPR-Cas system 7 (Figure 1). Two independently evolved families of type I-B Tn7-CRISPR-Cas elements also have two TniQ proteins in each element 3, 6 and seem likely to share a similar division of functions (Figure 1).…”

“…One branch of Tn7-like elements with the type I-F3 CRISPR-Cas system use an Xre-family protein for a form of zygotic induction to strongly induce the expression of a guide RNA specific to the chromosomal attachment site when they enter a new host ( rtaC in Figure 1). The att -targeting guide RNA is subsequently strongly repressed once the element is established in the host 7 . No known transcriptional regulators were found broadly conserved across Tn7-CRISPR-Cas12K elements in our analysis.…”

“…Of interest are multiple examples where CRISPR-Cas systems associate with a specialized type of transposons called Tn7-like elements 12, 13 . These natural systems of guide RNA-directed transposition show promise as tools for precision integration of genetic payloads, but fundamental questions remain with how they normally function and how they interact with canonical active CRISPR-Cas systems 4-7 .…”

“…In one well-represented group of Tn7-CRISPR-Cas elements functionally distinct guide RNAs allow two transposition pathways analogous to Tn7 7 . These elements evolved from the canonical I-F1 CRISPR-Cas systems, as a variant called I-F3 with Cas6, Cas7, and a fused version of Cas5 and Cas8 as an effector complex 3 (Figure 1).…”