Ben Kleinstiver

An inspirational panel this morning with none other than baby KJ himself - highlighting the stories of heroes @ahrensnicklas.bsky.social & @kiranmusunuru.bsky.social ( hosted by @jasonmast.bsky.social).

Crescendo: KJ's Dad on watching 🏈 with his son: "This is what we thought we would never get". ❤️👏

Congrats to @connorjtou.bsky.social on being named a 2025 #STATWunderkind. His enthusiasm in the lab is contagious and he thrives on doing really hard things.
Really well deserved recognition for an innovator and emerging leader in our field! 👏 🙌
@mgbresearch.bsky.social @harvardmed.bsky.social

Back in June, at our MSMDS conference, we shared how researchers were advancing their work on gene editing for ACTA2 R179H.

Today, we can celebrate that this effort is now peer-reviewed & published in @nature.com! 🎉
t.co/6WjTMRnAOi

#MSMDS #ACTA2 @markelindsay.bsky.social @bkleinstiver.bsky.social

Thanks, John! Hope all is well

Treatment of a severe vascular disease using a bespoke CRISPR–Cas9 base editor in mice Nature Biomedical Engineering - Engineering a mutant-specific customized base editor precisely corrects a mutation while minimizing bystander edits, leading to substantial phenotypic recovery in...

Please check out these links:
- free link to our manuscript: rdcu.be/eFAEz
- MGH press release: www.massgeneralbrigham.org/en/about/new...
- coverage in Fierce Biotech: www.fiercebiotech.com/research/cus...
- coverage in Precision Medicine Online: www.precisionmedicineonline.com/regulatory-n...

Together, our results demonstrate the value in using the mutant allele as a substrate in engineering campaigns, resulting in a bespoke enzyme with improved efficacy and safety compared to wild-type SpCas9 base editors.
🔧🧬 🚀

This was a wonderful collaboration and was funded by $$ from the NIH.

In vivo base editing via AAV9 or a smooth muscle cell tropic capsid AAV-PR (@casey-maguire-lab.bsky.social) substantially extended lifespan of treated MSMDS mice (+ key phenotypic changes). Notably, most AAV-ABE treated MSMDS mice died from bowel impaction, which should be avoidable in human.

@markelindsay.bsky.social and Dr. Musolino's labs created an MSMDS mouse model (that recapitulated many human phenotypes!), which we then utilized for in vivo experiments to deliver our optimized enhanced VRQR (eVRQR) ABE via a dual AAV approach. 🐁

We then performed a comprehensive off-target nomination workflow via an updated GUIDE-seq2 pipeline and the base editor-specific CHANGE-seq-BE assay, followed by rhAmpSeq-based validation of OTs (finding evidence of a small number of intergenic OTs).

To maximize on-target editing at this NGA PAM target site, we undertook a mutant allele-specific engineering approach to develop a bespoke enzyme with enhanced efficiency. Stacking activity-potentiating mutations with VRQR improved correction with minimal bystanders! 💯 ⬆️

To avoid this bystander we spatially minimized access to the M178 adenine by shifting the edit window of the ABE by 4 nt. Using either a PAM-relaxed SpG enzyme (@russelltwalton.bsky.social) or our more selective SpCas9-VRQR, this new target site dramatically minimized the M178V bystander! 🍻

By testing the impact of ACTA2 variant overexpression in human smooth muscle cells, we found that the M178V bystander edit resulted in disrupted actin polymerization; not as severely as the MSMDS R179H mutation, but this is a bystander edit that should be avoided. 🫨

When testing latest generation ABEs in an ACTA2 R179H cell line, we observed high levels of R179H correction (🎉!), but unfortunately most corrected alleles also contained a bystander edit that causes an ACTA2 M178V mutation (😩🤔). Does this bystander edit matter?

MSMDS is caused by a point mutation in the ACTA2 gene, which results in an R179H amino acid substitution - a mutation that should be correctable using adenine base editors (ABEs).

Multi-systemic smooth muscle dysfunction syndrome (MSMDS) is a rare a genetic vascular disease affecting children that Dr. Musolino sees in clinic, known to cause stroke, aortic dissection, and death. There are no therapies, motivating us to explore genome editing-based Tx for these patients.

This project has been a 5+ yr collaboration with Patricia Musolino and @markelindsay.bsky.social's groups, involving 30 co-authors! 🤝🙏🤲

Huge thanks to co-first authors Christiano Alves, Sabya Das, and Vijay Krishnan, and members from my group Leillani Ha, Hannah Stutzman, Lauren Fox, and others!

Treatment of a severe vascular disease using a bespoke CRISPR–Cas9 base editor in mice - Nature Biomedical Engineering Engineering a mutant-specific customized base editor precisely corrects a mutation while minimizing bystander edits, leading to substantial phenotypic recovery in mouse models of multisystemic smooth ...

Optimization of a bespoke base editor to treat a severe pediatric vascular disease! 🫀🧬
Our manuscript describes:
1️⃣ Engineering a target-specific BE🧬
2⃣ A *must avoid* bystander edit that occurs with WT SpCas9 BEs! 🙅‍♂️
3⃣ Extension of lifespan after in vivo editing! 🐁✅

www.nature.com/articles/s41...

Exciting to see our protein binder design pipeline BindCraft published in its final form in @Nature ! This has been an amazing collaborative effort with Lennart, Christian, @sokrypton.org, Bruno and many other amazing lab members and collaborators.

www.nature.com/articles/s41...

If you like transposons...
If you you love genome editing...
Or if you just like random bird animations,

we have the paper for you!

We (@kedmonds.bsky.social et al) are happy to share our work turning a songbird retrotransposon into a genome editing tool. 🐣 (1/n)

🚨 Our work on optimizing single-stranded DNA donors with Cas12a binding moieties is published at @moltherapy.bsky.social Nucleic Acids! 🧪

Since the preprint, we added two super interesting findings. Short 🧵👇 (1/5)

www.cell.com/molecular-th...

#ImmunoSky #GeneEditing #CARTcells

Less than one day to celebrate a major win for science, before returning to our regularly scheduled programming of destruction of the American scientific enterprise and capitulation..

1/10 Today in @science.org in collaboration with
the Liu group we report the development of a laboratory-evolved CRISPR-associated transposase (evoCAST) that supports therapeutically relevant levels of RNA-programmable gene insertion in human cells. drive.google.com/file/d/1I-Ub...

Genomes encode biological complexity, which is determined by combinations of DNA mutations across millions of bases

In new work @arcinstitute.org, we report the discovery and engineering of the first programmable DNA recombinases capable of megabase-scale human genome rearrangement

Baby Is Healed With World’s First Personalized Gene-Editing Treatment

A gene-editing treatment used on a 9½-month-old boy with a rare condition has the potential to help people with thousands of other uncommon genetic diseases #NBTNewsBeat www.nytimes.com/2025/05/15/h...

For more about how Rachel created this bespoke Cas9 protein, see here!

bsky.app/profile/bkle...

An incredible collaborative effort led by @kiranmusunuru.bsky.social @ahrensnicklas.bsky.social @urnov.bsky.social & others.
Congratulations to @rachelsilverstein9.bsky.social who designed the Cas9 enzyme that went into this drug.
Wishing K.J. & his family all the best 🙏

Today "a milestone in the evolution of personalized therapies for rare & ultra-rare inborn errors of metabolism"
—the 1st human to undergo custom genome editing
—from decades of NIH funded research
www.nejm.org/doi/full/10....
@nejm.org
www.nejm.org/doi/full/10....
www.nytimes.com/2025/05/15/h...

A screenshot of the termination notice showing "Outstanding Investigator Grants"

A screenshot of the termination notice with "This award is terminated effective the date of this award, due to unsafe antisemitic actions that suggest the institution lacks concern for the safety and wellbeing of Jewish students." highlighted

Yesterday, the NIH R35 “Outstanding Investigator” grant to fund scientists in my lab studying antibiotic resistance was terminated for reasons not related to the content of the science, or any actions taken by me or members of my lab

Plasmid of the Day

pCMV-T7-SpCas9(MKRCMV)-P2A-EGFP (AHK162)
Depositor: Benjamin Kleinstiver @bkleinstiver.bsky.social
Purpose: pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MKRCMV amino acid substitutions

www.addgene.org/223075/

Linking CRISPR–Cas9 double-strand break profiles to gene editing precision with BreakTag - Nature Biotechnology The genome-wide landscape of Cas-induced double-strand breaks and end structures is profiled at nucleotide resolution.

The genome-wide landscape of Cas-induced double-strand breaks and end structures is profiled at nucleotide resolution go.nature.com/44DO2Cc
rdcu.be/ekKtv