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!

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.

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 🤗)

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!