Stepwise DNA unwinding gates TnpB genome-editing activity
Zhou, Z.; Saffarian-Deemyad, I.; Shi, H.; Weiss, T.; ur-Rehman, M. M.; Vohra, K.; Skopintsev, P.; Yoon, P. H.; Trinidad, M. I.; Langeberg, C. J.; Kamalu, M.; Amerasekera, J.; Doherty, E. E.; Aris, K. D. P.; Al-Sayyad, N.; Thornton, B. W.; Weissman, R. F.; Wasko, K. M.; Esain-Garcia, I.; DeTurk, E. C.; Savage, D. F.; Jacobsen, S. E.; Bryant, Z.; Doudna, J. A.
Show abstract
TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through formation of a partially unwound intermediate state to a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in vivo. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.
Matching journals
The top 3 journals account for 50% of the predicted probability mass.
Similar papers in this journal
- Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells 98%
- Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch 97%
- Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules 97%
Similar papers in this journal
Similar papers in this journal
- UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay 97%
- Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors 97%
- Simultaneous measurement of biochemical phenotypes and gene expression in single cells 96%
"Similar papers" are the closest papers from that journal in the model's embedding space. They show what the match is built on, but the ranking comes mostly from a classifier over the whole training set, not from these examples alone.