Thanks! We looked at host response, and it's very similar to the immune response reported by Doudna lab (link below). That paper's title contains the phrase "minimally immunogenic Cas9 RNPs" promising for RNPs! I think RNPs' transient nature is a big advantage. pubmed.ncbi.nlm.nih.gov/37403358
Huge credit to @bmanohar.bsky.social for leading this collaborative effort, based at @innovativegenomics.bsky.social & @berkeleymcb.bsky.social, with major contributions from OSU's Krys Bankiewicz & Russ Lonser, as well as crucial support from NIH's SCGE consortium @scge.bsky.social
Together, these findings establish Neuro-PERC as a versatile, scalable, well-tolerated platform… as well as a promising candidate for clinical translation in neurotherapeutics. 🐭🐷🧠🧬✂️
Finally, we evaluated Neuro-PERC in a severe murine model of Huntington’s disease. Targeting of the human mutant HTT allele produced efficient on-target editing, reduced mutant HTT levels, and extended mouse survival relative to control mice.
Neuro-PERC achieved broad putaminal coverage with clear GFP loss (indicating successful editing) within the successfully targeted area. This confirmed that Neuro-PERC can be scaled to large-brained mammals using neurosurgical approaches already in clinical use.
To explore the translational potential of this approach, we performed MRI-guided CED into the putamen (a striatal region) of juvenile pigs at U of Missouri. This animal model allowed us to evaluate delivery across a brain volume substantially larger than that of the mouse. 🐷🧠
Using a dedicated reporter mouse, adenine base editor (ABE) delivered via Neuro-PERC mediated efficient A→G base editing in vivo, confirming compatibility with multiple CRISPR modalities beyond nuclease-mediated editing.
Neuro-PERC mediated robust in vivo editing of striatal neurons, achieving ~40–60% edited volumes with high reproducibility. Independent validation at @jacksonlab.bsky.social recapitulated results, even after our samples were shipped coast-to-coast.
To determine whether Neuro-PERC supports editing of neurons in vivo, we performed convection-enhanced delivery (CED) into the mouse striatum across multiple reporter models (Ai9, & GFP reporters activated by Cas9 nuclease or adenine base editor).🐭🧠
Neuro-PERC’s particles are ~20 nm, a small size that’s essential for effective spread through brain tissue. This delivery cocktail is compatible with sterile filtration & freeze/thaw. Our brain-ready formulation boosted editing in Ai9-derived NPCs relative to “classic” PERC.
We first evaluated Neuro-PERC in Ai9-derived mouse neural progenitor cells (NPCs). Peptide-enabled delivery markedly increased tdTomato reporter expression relative to Cas9 RNP alone, with peptides A5K & P55 supporting efficient editing without electroporation.
To improve neuronal delivery of CRISPR RNPs, Neuro-PERC combines pre-formed CRISPR enzymes (in RNP format) with endosome-escape peptides, resulting in a simple yet robust cocktail compatible with CNS applications. This builds on PERC, our previous technology.
Big news! Our team developed Neuro-PERC, a non-viral CRISPR delivery platform for genome editing of the brain. In our new preprint - bit.ly/NeuroPERC_pre - we report efficient editing in multiple models & species, including field-first results in large animals (pigs) 🧵👇
Thanks to Gen Eng News for the nice write-up on this work, which is available here: bit.ly/GEN-hiNLS
We put our two favorite hiNLS constructs on
@addgene.bsky.social for anyone interested in giving them a try:
www.addgene.org/search/catal...
What did we see?
• hiNLS constructs supported high-yield production
• hiNLS can improve genome editing efficiency
• this was true via 2 delivery methods:
electroporation & PERC (peptide-mediated)
• more NLS is better, but only up to a point
• some NLS can pack more of a punch: cMyc > SV40
To minimize risks of diminished protein production yields associated with N/C-terminal tags, Eric put "hairpin" pairs of NLS into the Cas9 protein backbone. If you like files *in* the computer, you'll love NLS *in* the Cas9. We call this layout hiNLS (hairpin/internal NLS).
NLS has always been an important contributor to genome editing, but we wondered if more NLS = higher editing efficiency. Prior work from Scot Wolfe, Dan Bauer, Junwei Shi, and others suggested this may be the case, but we wanted to take things a step further, and it yielded surprising results!
Cas9 gets an upgrade! Check out our new paper in The CRISPR Journal: bit.ly/hiNLS-Cas9 Eric Noel (who is on the job market!) created Cas9 constructs w/ nuclear localization signals (NLS) in the backbone, boosting genome editing activity in T cells, as seen by editor @srishtisahu.bsky.social #CRISPR
Teddy is truly a local hero, for so many reasons!
Massive credit to stellar co-first authors Srishti @srishtisahu.bsky.social & Madalena "Madi" Castro, as well as our wonderful collaborators (David Nguyen, Joe Muldoon, and Justin @j-eyquem.bsky.social). In case you need access to the PDF, I believe this link should work: rdcu.be/ebZFu
We also built an FAQ page with some complementary resources, including recommendations on sourcing protein & peptide. We will continue updating this page with new information. Check it out!
www.rosswilsonlab.org/perc
This paper is incredibly detailed and should provide the information you need to get PERC working well for RNP delivery ex vivo using Cas9 or Cas12a. The big idea is to omit the electroporator and instead mix your RNP enzyme with an inexpensive and easy-to-use peptide.
I'm proud to share our protocol on CRISPR enzyme delivery in primary human cells using PERC, a non-viral & hardware-independent technology. We previously described PERC in T cells, and here we extend its use to hematopoietic stem/progenitor cells (HSPCs).
www.nature.com/articles/s41...
Credit goes to everyone who contributed: Srishti Sahu, Lorena de Oñate, Bruno Solano, and especially lead author Christy George, who worked tirelessly on this review.
Ex vivo therapies have predominated so far because they sidestep the substantial challenges of in vivo delivery. As potent as cell therapies can be, transplant can limit access and delay treatment. Fortunately, off-the-shelf T cell therapies and in vivo delivery are poised to change this landscape.
Therapeutic genome editing of hematopoietic stem cells (HSCs) and T cells has been getting better and better over the last ~15 years, with progress greatly accelerated by the advent of CRISPR. Here's our timeline figure in presentation-friendly "landscape" format (an online exclusive 🤗)
I'm proud to share our review on CRISPR therapies for the blood. It has been about a year since Casgevy was approved for treatment of sickle cell disease, and edited CAR-T cells are showing more and more promise. Key figures shared below; paper is open access here: www.liebertpub.com/doi/full/10....
The Wilson lab has two new PhD candidates: congrats to Brigette Manohar and Christy George on passing their qualifying exams in the Cal MCB program! They're both working to improve CRISPR delivery, potentially enabling new therapies for the central nervous system.
If we're looking at, say... the average weight of an animal, volume of a cell, odds of getting cancer, half-life of a protein... any of those seems like a big deal!
Excited for you - congrats!