Ben Kleinstiver
@bkleinstiver.bsky.social
Associate Professor @ Mass General Hospital & Harvard Medical School
Genome editing / Protein eng. / Molecular medicine 🇨🇦🧬
Kayden-Lambert MGH Research Scholar '23-28
Genome editing / Protein eng. / Molecular medicine 🇨🇦🧬
Kayden-Lambert MGH Research Scholar '23-28
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". ❤️👏
Crescendo: KJ's Dad on watching 🏈 with his son: "This is what we thought we would never get". ❤️👏
October 16, 2025 at 12:15 AM
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". ❤️👏
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
Really well deserved recognition for an innovator and emerging leader in our field! 👏 🙌
@mgbresearch.bsky.social @harvardmed.bsky.social
October 16, 2025 at 12:07 AM
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
Really well deserved recognition for an innovator and emerging leader in our field! 👏 🙌
@mgbresearch.bsky.social @harvardmed.bsky.social
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.
September 12, 2025 at 2:00 AM
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. 🐁
September 12, 2025 at 2:00 AM
@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).
September 12, 2025 at 2:00 AM
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! 💯 ⬆️
September 12, 2025 at 2:00 AM
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! 🍻
September 12, 2025 at 2:00 AM
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. 🫨
September 12, 2025 at 2:00 AM
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?
September 12, 2025 at 2:00 AM
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).
September 12, 2025 at 2:00 AM
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.
September 12, 2025 at 2:00 AM
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.
Then, with Qin Liu @MassEyeAndEar, we utilized PAMmla to develop bespoke allele-selective Cas9 nucleases to treat a blindness causing mutation.
Importantly, PAMmla reversed the biology of Cas9, now able distinguish against a canonical NGGG PAM & instead effectively target an NGTG PAM.
Importantly, PAMmla reversed the biology of Cas9, now able distinguish against a canonical NGGG PAM & instead effectively target an NGTG PAM.
April 22, 2025 at 8:57 PM
Then, with Qin Liu @MassEyeAndEar, we utilized PAMmla to develop bespoke allele-selective Cas9 nucleases to treat a blindness causing mutation.
Importantly, PAMmla reversed the biology of Cas9, now able distinguish against a canonical NGGG PAM & instead effectively target an NGTG PAM.
Importantly, PAMmla reversed the biology of Cas9, now able distinguish against a canonical NGGG PAM & instead effectively target an NGTG PAM.
To enable users to predict their own bespoke Cas9 enzymes, Rachel developed an in silico directed evolution pipeline and worked with @lucapinello.bsky.social's lab to build a website that permits prediction and navigation of novel Cas9 proteins *in real time* - check it out!
pammla.streamlit.app
pammla.streamlit.app
April 22, 2025 at 8:57 PM
To enable users to predict their own bespoke Cas9 enzymes, Rachel developed an in silico directed evolution pipeline and worked with @lucapinello.bsky.social's lab to build a website that permits prediction and navigation of novel Cas9 proteins *in real time* - check it out!
pammla.streamlit.app
pammla.streamlit.app
Working with Dr. Suk See De Ravin @NIH, we demonstrated that PAMmla enzymes are highly effective as base editors to correct mutations in patient-derived primary cells while also minimizing off-targets.
No need to use PAM-relaxed enzymes like SpG anymore - use more specific PAMmla proteins instead!
No need to use PAM-relaxed enzymes like SpG anymore - use more specific PAMmla proteins instead!
April 22, 2025 at 8:57 PM
Working with Dr. Suk See De Ravin @NIH, we demonstrated that PAMmla enzymes are highly effective as base editors to correct mutations in patient-derived primary cells while also minimizing off-targets.
No need to use PAM-relaxed enzymes like SpG anymore - use more specific PAMmla proteins instead!
No need to use PAM-relaxed enzymes like SpG anymore - use more specific PAMmla proteins instead!
Because PAMmla-predicted enzymes are more selective for their PAMs, they encounter fewer off-target sites around the genome.
This results in safer enzymes that minimize unwanted off-target editing.
This results in safer enzymes that minimize unwanted off-target editing.
April 22, 2025 at 8:57 PM
Because PAMmla-predicted enzymes are more selective for their PAMs, they encounter fewer off-target sites around the genome.
This results in safer enzymes that minimize unwanted off-target editing.
This results in safer enzymes that minimize unwanted off-target editing.
Testing PAMmla enzymes in human cells led to encouraging results - as we'd hoped, PAM altered enzymes enabled higher levels of on-target editing for nucleases and base editors, while minimizing editing at unwanted PAMs in ways previously not possible with PAM-relaxed enzymes.
April 22, 2025 at 8:57 PM
Testing PAMmla enzymes in human cells led to encouraging results - as we'd hoped, PAM altered enzymes enabled higher levels of on-target editing for nucleases and base editors, while minimizing editing at unwanted PAMs in ways previously not possible with PAM-relaxed enzymes.
We validated that PAMmla could accurately predict the PAM profiles of Cas9 enzymes, both previously unseen before in nature or in our training data sets, and those of previously engineered SpCas9 PAM variant enzymes (developed by us or others).
April 22, 2025 at 8:57 PM
We validated that PAMmla could accurately predict the PAM profiles of Cas9 enzymes, both previously unseen before in nature or in our training data sets, and those of previously engineered SpCas9 PAM variant enzymes (developed by us or others).
@rachelsilverstein9.bsky.social then utilized machine learning to investigate all of these proteins including the rarer enzymes, training a PAM machine learning algorithm (PAMmla) that could then predict the PAM requirements of all 64 million Cas9 proteins varied at these 6 amino acids.
April 22, 2025 at 8:57 PM
@rachelsilverstein9.bsky.social then utilized machine learning to investigate all of these proteins including the rarer enzymes, training a PAM machine learning algorithm (PAMmla) that could then predict the PAM requirements of all 64 million Cas9 proteins varied at these 6 amino acids.
The selections yielded two major phenotypes: enzymes that can target 'NGG' sequences (akin to wild-type SpCas9), or those that can tolerate a relaxed 'NGN' PAM (similar to previously developed generalist enzymes). But hidden in this dataset were some more rare Cas9s enzymes..
April 22, 2025 at 8:57 PM
The selections yielded two major phenotypes: enzymes that can target 'NGG' sequences (akin to wild-type SpCas9), or those that can tolerate a relaxed 'NGN' PAM (similar to previously developed generalist enzymes). But hidden in this dataset were some more rare Cas9s enzymes..
We performed saturation mutagenesis of 6 amino acid residues in Cas9, subjected this library of enzymes to a large selection experiment (against different PAMs), and then utilized scalable assays to sequence & characterize ~800 Cas9 proteins (thx to HT-PAMDA; @russelltwalton.bsky.social )
April 22, 2025 at 8:57 PM
We performed saturation mutagenesis of 6 amino acid residues in Cas9, subjected this library of enzymes to a large selection experiment (against different PAMs), and then utilized scalable assays to sequence & characterize ~800 Cas9 proteins (thx to HT-PAMDA; @russelltwalton.bsky.social )
Protein engineering workflows can result in generalist enzymes due to limitations in scale or a lack of assays to characterize enzymes. While generalist enzymes can be useful for research, designer enzymes may be better fit for therapeutics with advantages in safety & efficacy.
April 22, 2025 at 8:57 PM
Protein engineering workflows can result in generalist enzymes due to limitations in scale or a lack of assays to characterize enzymes. While generalist enzymes can be useful for research, designer enzymes may be better fit for therapeutics with advantages in safety & efficacy.
Major kudos to Cas9 engineer extraoirdinare @rachelsilverstein9.bsky.social who bravely led this project from her Harvard BBS Program rotation until now.
A massive collaborative effort including contributions from Nahye Kim @ann-sophiekroell.bsky.social @russelltwalton.bsky.social & many others.
A massive collaborative effort including contributions from Nahye Kim @ann-sophiekroell.bsky.social @russelltwalton.bsky.social & many others.
April 22, 2025 at 8:57 PM
Major kudos to Cas9 engineer extraoirdinare @rachelsilverstein9.bsky.social who bravely led this project from her Harvard BBS Program rotation until now.
A massive collaborative effort including contributions from Nahye Kim @ann-sophiekroell.bsky.social @russelltwalton.bsky.social & many others.
A massive collaborative effort including contributions from Nahye Kim @ann-sophiekroell.bsky.social @russelltwalton.bsky.social & many others.
