Ross Wilson

New Strategy Enhances CRISPR Editing Efficiency for Therapeutic Use Latest study leverages new constructs for nuclear localization signal (NLS) sequences to improve editing efficiency in human immune cells.

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

a man and a woman are standing next to each other in a room with a painting on the wall ALT: a man and a woman are standing next to each other in a room with a painting on the wall

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!

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells Nature Protocols - Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) uses an amphiphilic peptide reagent to mediate intracellular delivery of CRISPR enzymes for...

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.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells - Nature Protocols Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) uses an amphiphilic peptide reagent to mediate intracellular delivery of CRISPR enzymes for high-efficiency editing of stimulat...

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 🤗)

Genome Editing Therapy for the Blood: Ex Vivo Success and In Vivo Prospects | The CRISPR Journal Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorde...

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!

Congrats to Karikó and Weissman on the Nobel for mRNA modifications! Their work was not appreciated by the university or reviewers at the time, but has saved millions of lives. A great example of the value of discovery science and reminder to not limit academic research to immediate applications.