Excited that our paper is now online @Nature! Selective TnsC recruitment enhances the fidelity of RNA-guided transposition - the result of a fantastic collaboration of the @shsternberg and @IsraelF96135088 labs. (nature.com/articles/s41586-0…) A thread:
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We previously showed that type I-F CRISPR-associated transposons (CASTs) facilitate programmable RNA-guided DNA integration - without DSBs. The type I-F CAST system (Tn6677) is highly accurate, allowing precise insertion of large DNA payloads for genome engineering.
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Together with Leslie Beh, we used ChIP-seq in E. coli to resolve how recruitment events of individual Cas and transposon (Tn) proteins conspire in vivo to achieve this remarkably targeted DNA insertion behavior.
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We found that initial target site selection by Cascade is very promiscuous: Cascade samples hundreds of off-targets across the genome - a surprising result since we routinely observe >99% of integration events to occur at the target site. Most off-targets resemble the crRNA.
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Given its important role in target site selection, @minjookies purified the challenging TnsC and the amazing @IsraelF96135088 (make sure to check out his thread) built the beautiful cryoEM structures of heptameric TnsC ring complexes. TnsC oligomerization is ATP-dependent.
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Aug 24, 2022 · 10:45 PM UTC
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The pore’s dimensions seemed ideal to accommodate dsDNA. @minjookies mastered the purification DNA-bound TnsC, for which @IsraelF96135088 refined not one but two DNA-bound structures. The overall heptameric structures of DNA-free and DNA-bound TnsC are very similar. But…
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However, the TnsC ring slightly opens upon DNA binding, forming a small cleft between adjacent protomers. We believe this intersubunit flexibility may allow loading of TnsC heptamers onto DNA. Thanks to @IsraelF96135088 for highlighting the differences in the video.
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We propose a Cascade-TniQ-TnsC structural model explaining the ~50 bp spacing between the crRNA target sequence and integration site. Overall, comparable to a funnel, each protein recruitment step in type I-F RNA-guided transposition adds a layer of selectivity.
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This project was only possible because of the expertise of many amazing scientists. A big thank you @shsternberg, @IsraelF96135088, @minjookies, Leslie, @JingWang0610, @Vopinator, @tehhoyen, @jerrintgeorge, @jason_mohabir, @AcreeChris! I could not wish for a better team!
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