Karl opined that there may be an evolutionary reason for this feeling, because we are a social species and survival depended on teaching. Now we just need to find the pathway from the cortex to the VTA for that.

Celebration at Stanford Bioengineering for Karl Deisseroth’s Nobel Prize. His commitment to teaching came up, and Karl said something very interesting: that one of the best feelings is finishing a classroom session and thinking it went well, that the students learned something. Completely agree!

And would not have been possible without early support from Stanford Bio-X, the Focused Ultrasound Foundation, and the SMA Foundation, and sharing the same roof with the Hong lab at the Stanford Wu Tsai Neuroscience Institute

Dongin Lee and Hanlee Ji for NGS; Nicole Repina and David Schaffer for engineering a programmable multi-well illuminator; and of course Guosong Hong for co-directing the project!

Many people to thank on this fun and intense collaborative project: Michael Westberg for ideas on designs, Yichi Su for the reporter construct; Marigold Malinow, Xiang Wu, Shan Jiang, and Sa Cai for MLNT synthesis and ultrasound experiments, Dongyun Jiang for histology...

There is more to explore, e.g. cell-type-specific expression and generalizing ps-Cas12f to non-editing outputs. More broadly, we hope that remote control of engineered proteins in vivo inspires others to think of other ways in which engineering can lead to smarter medicines.

Actually we found Cas12f variants to have less off-target activity than Cas9, replicating earlier results. So we are taking a low risk and making it even lower. IMO all future CRISPR will be Cas12f CRISPR: smaller, more specific, and just as active — and now regulatable in 4D.

So why is local gene editing useful? For example to treat Huntington's Disease, we'd prefer to disrupt HTT in just the striatum if possible, in case it has essential functions elsewhere. Also caging Cas12f by default reduces off-target toxicity that would otherwise accumulate.

It's interesting to think about what's happening here: The MLNTs circulate in the bloodstream. The light they produce cross the BBB to activate ps-CasMINI whose genetic instructions had crossed the BBB earlier in AAV!

So we delivered ps2-CasMINI across the BBB using AAV serotype CAP.B10 (from the Gradinaru lab), then perform local ultrasound induction of reporter gene editing at one location in the brain. (We show the pseudocolor overlays because that's standard but we like to show raw luminescence images too)

The brain is one place where local gene editing would be useful. As neurologists know, many conditions are localized, so it would be nice if CRISPR-based genetic treatments can be local too. But location-specific promoters often don't exist, and it would be nice to avoid surgery.

And so in a close collaboration with the Hong lab, we injected AAV to express ps-Cas12f in muscle, then injected mechanoluminescent nanotransducers (MLNTs), then performed ultrasound to trigger light production at the focal point and induce Cas12f-mediated gene editing.

Okay but external illumination isn't really local or deep. That's where the 2nd engineering effort comes in, in the development of mechanoluminescent nanoparticles by Guosong Hong's lab. These are particles that generate cyan light upon ultrasound stimulation, perfect for pdDronpa!

And it works in vivo! We delivered each system in a single AAV to mouse legs, then applied external light Light induced luciferase transgene activation (due to gRNA-targeted editing of a stop signal in front of the luciferase) by ~19x vs no light, reaching similar activity levels as unfused CRISPR

We fixed this. We engineered a faster-switching pdDronpa2, then made "ps2" variants with newer higher-activity Cas12f variants, eCas12f and AsCas12f-HKRA The result was ~20x more gene editing in response to lower amounts of light, reaching the same activity as unfused Cas9.

But pdDronpa's photoresponse was fairly inefficient, occurring in <1% of photon absorbance events (the other 99% producing fluorescence). Thus psCasMINI needed more photons to switch vs. natural photosensory domains, e.g. LOV or cryptochrome. And CasMINI was not as active as the standard Cas9.

Amazing postdoc Daesun Song took up the project. He quickly made a psCasMINI that showed ~13-fold induction of gene editing with light, and induced activity similar to unfused CasMINI. All within 2.5kB, small enough to fit, along with a gRNA gene, into a single AAV particle

But the cool thing is: in the Cas12f:gRNA complex, the two N-termini of Cas12f already flank the gRNA. Thus if we fuse just *1* pdDronpa domain to the Cas12f NT, the pdDronpa dimer should form across the gRNA to cage it Here's my manual model from 2021. No AF, no ML. Just PyMol.