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Mechanism of target site selection by type V-K CRISPR-associated transposases by Jerrin Thomas George; Christopher Acree; Jung-Un Park; Muwen Kong; Tanner Wiegand; Yanis Luca Pignot; Elizabeth H. Kellogg; Eric C. Greene; Samuel H. Sternberg is a Medicine article available to read on EtoBox.

What is Mechanism of target site selection by type V-K CRISPR-associated transposases about?

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, “untargeted” transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo–electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

Who reads Mechanism of target site selection by type V-K CRISPR-associated transposases?

Author

Jerrin Thomas George; Christopher Acree; Jung-Un Park; Muwen Kong; Tanner Wiegand; Yanis Luca Pignot; Elizabeth H. Kellogg; Eric C. Greene; Samuel H. Sternberg

Publisher

American Association for the Advancement of Science (AAAS)

Published

2023

Language

EN

Field

Medicine (Social Sciences)