Super excited to share our pre-print and my first ‘first author’ work: “Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection.” @ucsdbiosciences @hhminews.🧵1/.

RT @VirusOfMicrobes: Chase Morgan kicking of this session with the molecular transmission of proteïns through the replication nucleus of gi….

Congrats, Chase! 🙌.

Congratulations, @owen_t_tuck, @ben_a_adler and team! 🙌🎉.

Eray is killin it out here. Congrats!! 🙌.

Last but not certainly not least, thanks to @poglianojoe for his endless patience and support and the whole Pogliano team - I could not ask for a better lab fam. ❤️.

Especially big shout out and thank you to former undergrad in the lab and current @RockefellerUniv PhD student @jina_leemon, aka the Goslar whisperer. Many late nights and burritos later, we made it!.

Additional special thanks to @3rdFloorTom, Emeric Charles, @dfsavage, @ben_a_adler and @bradyfcress for helping us bring CRISPRi-ART to the lab and members of the @hhminews Emergent Pathogens Initiative phage consortium for their support and feedback 12/.

Huge thanks to authors @jina_leemon,@joshhutchings94,@arica_vanderwal, @enustuneray,@ben_a_adler,@annie_biotics,@iamamardeep,@zaidita_k0430,@chmorgan91,@GhassemianMajid, Emeric Charles,@bradyfcress, @dfsavage,@doudna_lab,@kit_ucsd,@_KevinCorbett, @thevillalab & @poglianojoe 11/.

Time to update our model of the Chimallviridae life cycle! Upon injection, the viral genome is enclosed within the EPI vesicle (likely formed from host inner membrane), protecting it until formation of the ChmA phage nucleus, at which point genome replication begins. 10/

But also! These early phage infection (EPI) vesicles, were actively expressing phage genes transcribed INSIDE the vesicle, with the mRNA exported and translated as shown by the polysomes extending from the vesicle surface and confirmed by mass spec. 9/

Using cryo-ET, we took an even closer look at the arrested phage genome and made an incredible discovery. The injected phage genomes were all enclosed in membrane-bound vesicles, previously suspected to be and now confirmed to be a pre- phage nucleus stage in the life cycle. 8/.

But what’s actually happening to Goslar when ChmA is gone? As always, we looked under the microscope to find out! Turns out that knocking down ChmA arrested Goslar infection at an early stage and effectively blocked all replication of the viral DNA. 7/

Using CRISPRi-ART, we determined that specific, targeted knockdown of ChmA severely inhibited reproduction of E. coli Chimalliviridae phage Goslar. 6/

CRISPRi-ART uses dCas13d to inhibit gene expression by targeting mRNA, so it can be used as a genetics tool for nucleus-forming phages as the viral mRNA must be exported from the nucleus for translation by host ribosomes. 5/

To overcome this barrier, we joined forces with the amazing @ben_a_adler and @bradyfcress. They, alongside Muntathar Al-Shimary, led the charge developing the incredibly powerful CRISPRi-ART technology, pre-printed earlier this week (hyyypee!) 4/.

However, it has remained unknown whether the phage nucleus is actually required for the viral life cycle. This protective barrier also shields the phage DNA within from genome editing tools, making it extremely difficult to tackle this key question. 3/.

Many eukaryotic viruses generate dedicated intracellular compartments for genome replication, but what about phages? Well, Chimalliviridae phages build a protective shell, the phage nucleus, out of the viral protein ChmA to enclose their replicating genomes during infection. 2/

Congratulations, @ben_a_adler, Muntathar, @bradyfcress and the rest of the team on this super exciting work! 🙌 Can't wait to see what exciting phage secrets CRISPRi-ART will unveil! 😉.

I'm very late to the hype train (🙈), but congratulations, @EnustunEray!! 🎉 So exciting to see and start getting to know this "rogues' gallery" of ChmA's interaction partners. 🦹🦹‍♂️🦹‍♀️.