Thank you Benjamin 😊

Thanks a lot Graham, I am looking forward to any question that may come up and hope that you and your lab enjoy the read 🙏

Thank you Kumar, this means a lot 🤩

Thanks a lot, hope you like where the story went since you last saw it in Sounio!

Thanks Marcel, I hope you like the paper :)

I hope we will get another chance for collaboration, this was great!! 💪

I am sure we will! Thank you for helping me refine this tweetorial 💪

Shoutout to @zonderlandg.bsky.social and our fantastic collaborators from @grothlab.bsky.social @biranalva.bsky.social, Nicolas Alcaraz, Alice Wenger and @nilskrietenstein.bsky.social, Irene Mordini, who took our discoveries to the next level, great team effort 🙌🙌🙌#ScienceResearch 12/12

Can a repaired locus still do its job? We challenged DSB-recovered cells with growth factor signaling.
Live-cell imaging revealed a 𝗯𝗹𝘂𝗻𝘁𝗲𝗱 𝗰-𝗠𝗬𝗖 𝗿𝗲𝘀𝗽𝗼𝗻𝘀𝗲 𝘁𝗼 𝗘𝗚𝗙 — both short- and long-term induction were defective. 11/n

Are 3D chromatin changes after DNA repair tied to 𝘄𝗲𝗮𝗸𝗲𝗻𝗲𝗱 𝗥𝗡𝗔 𝗵𝘂𝗯𝘀?
Using RNA FISH at the c-MYC locus, we found they are — local RNA compartments stay compromised even after repair, mirroring structural chromatin changes. 10/n

𝗔𝗿𝗲 𝟯𝗗 𝗰𝗵𝗿𝗼𝗺𝗮𝘁𝗶𝗻 𝗰𝗵𝗮𝗻𝗴𝗲𝘀 𝗮 𝗵𝗲𝗿𝗶𝘁𝗮𝗯𝗹𝗲 𝗰𝗼𝗻𝘀𝗲𝗾𝘂𝗲𝗻𝗰𝗲 𝗼𝗳 𝗗𝗡𝗔 𝗿𝗲𝗽𝗮𝗶𝗿?Labelling both ends of the c-MYC TAD revealed a stable increase in probe distance. @nilskrietenstein.bsky.social took a closer look with Region-Capture Micro-C and found heritable 3D structural changes in the entire c-MYC TAD! 9/n

𝗜𝘀 𝘁𝗵𝗲 𝗶𝗺𝗽𝗮𝗶𝗿𝗺𝗲𝗻𝘁 𝘁𝗲𝗺𝗽𝗼𝗿𝗮𝗿𝘆? Not at all. Even after >two rounds of replication, c-MYC TAD genes stay repressed after a single DSB!
And our collaborators @grothlab.bsky.social took it further — editing the Mcm2 locus in mESCs showed deregulation inherited over multiple passages. 8/n

𝗛𝗼𝘄 𝗺𝘂𝗰𝗵 𝗰𝗵𝗿𝗼𝗺𝗮𝘁𝗶𝗻 𝗶𝘀 𝗮𝗰𝘁𝘂𝗮𝗹𝗹𝘆 𝗮𝗳𝗳𝗲𝗰𝘁𝗲𝗱 𝗯𝘆 𝗮 𝘀𝗶𝗻𝗴𝗹𝗲 𝗗𝗦𝗕? As a first surprise, a single Cas9-induced DSB anywhere in the c-MYC TAD (>2.5 MB) affects local gene expression! Even when most of the cells have completed DNA repair and the break was over a megabase distant from the c-MYC ORF! 7/n

We used the >2.5 MB c-MYC TAD as our model targeting it by single Cas9 cuts, across the entire TAD. Next, we measure the c-MYC protein levels during and after DNA repair (marked by 53BP1 at the DSB) using high content single-cell microscopy of thousands of cells to generate population data. 6/n

These findings shift how we think about ageing, cancer, and the hidden “cost” of genome integrity.
It also raises big questions👇
• Which genes are most vulnerable to chromatin fatigue?
• How does chromatin refold after repair —and why isn’t it always perfect?
• How can we safely use CRISPR/Cas9? 5/n

IN SUM, post-repair chromatin shows deregulated genes, altered 3D structure, impaired RNA compartments and gene induction. IMPORTANTLY, all is inherited by daughter cells, so we term this post-repair state “𝗰𝗵𝗿𝗼𝗺𝗮𝘁𝗶𝗻 𝗳𝗮𝘁𝗶𝗴𝘂𝗲”, the 𝗰𝗼𝘀𝘁 𝗼𝗳 𝗗𝗡𝗔 𝗿𝗲𝗽𝗮𝗶𝗿 enabling survival and proliferation. 4/n

BACKGROUND: during DNA double-strand break (DSB) damage response and repair, large (>Mb) chromatin regions surrounding the DSB are modified and reshaped. The big Q: 𝗖𝗮𝗻 𝗰𝗵𝗿𝗼𝗺𝗮𝘁𝗶𝗻 𝗳𝘂𝗹𝗹𝘆 𝗿𝗲𝗰𝗼𝘃𝗲𝗿 𝗳𝗿𝗼𝗺 𝘁𝗵𝗲𝘀𝗲 𝗹𝗮𝗿𝗴𝗲-𝘀𝗰𝗮𝗹𝗲 𝗰𝗵𝗮𝗻𝗴𝗲𝘀 𝘄𝗶𝘁𝗵𝗼𝘂𝘁 𝗰𝗼𝗻𝘀𝗲𝗾𝘂𝗲𝗻𝗰𝗲 𝘁𝗼 𝗰𝗲𝗹𝗹𝘂𝗹𝗮𝗿 𝗽𝗵𝘆𝘀𝗶𝗼𝗹𝗼𝗴𝘆? 3/n

WHY? Targeted cleavage of DNA is routinely used for experimental and therapeutical genome editing ✂️ 🧬 But we show long-term consequences of CRISPR-Cas9 go beyond mutations and challenge the assumption that mutations are the only consequence of DNA breaks. 2/n