Lab News

Cas9 is a great DNA cutting enzyme, but it’s also a little weird. Unlike other nucleases (such as restriction enzymes), S, pyogenes Cas9 sticks on DNA for a looooong time. In fact, it spend the same amount of time on DNA whether it is active or inactive! In a test tube, it takes hours and hours to let go of even a cleaved DNA strand. So how does genome editing even work? Does a human cell care that Cas9 it stuck on its genome? Does it have ways to knock it off?

Superstar grad student Alan Wang’s new paper, out today in Molecular Cell, solves this mystery. Previous work suggested that RNA polymerase II was capable of displacing Cas9 in vitro. But Cas9-based technologies work even when targeted to a non-transcribed region. Alan’s first hint that something interesting was going on came from work in Xenopus egg extract, in collaboration with the lab of Johannes Walter. This “frog juice” is very often used to study DNA repair and can be elegantly deconstructed to figure out biological function. Cas9 in buffer sticks on DNA for a long time, but in Xenopus extract it comes off almost immediately!

Alan took an unbiased approach to figure out what was removing Cas9 from DNA. He fused recombinant Cas9 with a promiscuous biotin ligase, bound purified Cas9-ligase  to a plasmid, and used mass spectrometry to figure out what pushed the Cas9 off the plasmid. We were pleasantly surprised to find that both subunits of a dimeric histone chaperone called FACT were top mass spec hits! Follow-up experiments showed that FACT was necessary and sufficient for displacing Cas9 from DNA substrates. FACT was responsible for turning Cas9 from a multi-turnover “classic” nuclease enzyme into a single-turnover sticky enzyme!

In human cells, FACT had several interesting effects on genome manipulation. Knocking down FACT delayed homology directed repair and altered the balance of repair outcomes. FACT knockdown increased epigenetic marking from both CRISPRi and CRISPRa constructs, and increased CRISPRi phenotypes. We attribute this to increasing the residence time at a target site: giving the effect fused to Cas9 more time to have an effect on the genome. 

The take-home is that cells are not passive players, and play a leading role in genome manipulation. The cell is just as important as the enzyme! Alan’s work starts to reveal how cells monitor their genomes during Cas9 interventions.

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Welcome to Lena Kobel, who joins the lab as a Cell Line Engineer. Lena has a long history in genome engineering, with previous experience in Martin Jinek’s lab and at Caribou Biosciences. Lena will be working on precision cell models and screens to study the genetics of DNA damage and genome editing.

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Did you know that cells eat their own organelles? This is best known when damaged mitochondria are degraded by autophagy (aka mitophagy). Failure to perform mitophagy can lead to diseases such as Parkinson’s. But many other organelles are also degraded by autophagy. We have been studying autophagy of the endoplasmic reticulum (ER-phagy), which is much less understood than mitophagy. Engulfing a mitochondria in an autophagosome sounds pretty straightforward, but ends up being complicated. Now imagine needing to do that for one part of the ER network! A handful of direct ER-phagy receptors are known. But these receptors are always on the ER, so it was not clear what really initiates and controls ER-phagy. How does the cell know what part to engulf? What are the signals that turn this on and off? What happens when it goes wrong?

Superstars Amos Liang (postdoc) and Emily Lingeman (PhD student) tackled this question in a big way. Using a highly sensitive fluorescent reporter for ER-phagy, they used CRISPRi to ask what genes regulate ER-phagy. The first surprise was that intact mitochondrial oxidative phosphorylation is required to successfully initiate ER-phagy. This is odd because preventing oxidative phosphorylation actually initiates bulk autophagy. But the opposite is true for ER-phagy! The second big surprise was that a weird post-translational modification called UFMylation is required for ER-phagy. Lots of mechanistic work showed how UFMylation machinery is brought to the ER surface and what gets UFMylated during ER-phagy. There are some very interesting parallels to mitophagy, but using totally different machinery. Third, many of the genes involved in ER-phagy are involved in peripheral neuropathy in humans. Since their role in ER-phagy wasn’t previously known, it wasn’t understood how they were connected to cause human disease. This work suggests that failure to do ER-phagy links them all and leads to neurodegeneration. There’s a lot going on here, so read the paper to find out more.  Congrats to Amos and Emily!

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Genome editing is all about DNA repair. So if you want to get your cells to do more of what you want (*cough* HDR *cough), you’d better know how they make decisions about DNA repair pathways. To help people get the lay of the land, Ph.D. student Charles Yeh and former postdoc Chris Richardson wrote a review all about manipulating DNA repair decisions to influence genome editing outcomes. Out now in Nature Cell Biology!

Be sure to check out Table 1 for a very thorough, non-redundant summary of all the ways people have tried to redirect CRISPR-Cas repair in human cells!

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Cells have many ways to figure out that something is wrong. One of these is cGAS, which makes a cyclic dinucleotide (CDN) to activate STING innate immune signaling. CDNs are made during bacterial infection and tumor progression, and CDN derivatives are in development to re-activate immune cells next to a tumor. But how do CDNs secreted into the environment get into a target cell? This was the question asked by David Raulet’s lab, who collaborated with us on a genome-wide CRISPRi screen to find the CDN transporter. We helped the Raulet lab identify the folate transporter (SLC19A1) as a CDN importer. The Raulet lab plus further collaboration with Joshua Woodward’s lab figured out the mechanism. Lingyin Li’s lab also identified the folate transporter in a parallel collaboration with Mike Bassik, reported in Molecular Cell. Congrats to former lab members Benjamin Gowen and Stacia Wyman, who were authors on the paper, now out in Nature!

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Welcome to Zac Kontarakis, who is the head of the new Genome Engineering and Measurement Lab (GEML). The GEML is a new hub, jointly developed by Jacob Corn and the Functional Genomics Center Zurich, that will focus on the development of innovative approaches to genome engineering and their deployment to the Zurich research community. You may know Zac’s work already from his mind-blowing papers on mechanisms of genetic compensation. Stay tuned for more from Zac and the GEML!

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Matthias is a new postdoc in the lab, having joined after completing his Ph.D. with Johannes Zuber at the IMP in Vienna. Welcome!

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Three people joined the lab, all in one day! Erman is a postdoc, interested in DNA repair and genome editing, Kinga is our new lab manager, and will be keeping us all in line. Markus is a bioinformatician, working on quantifying editing outcomes from complex datasets. Welcome to the lab!

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The lab’s manuscript on DISCOVER-seq is out today in Science. DISCOVER-seq is a way to watch Cas enzymes doing their things in any cell, or even an organism. It uses recruitment of DNA repair factors to find off-targets and provides single-nucleotide resolution of Cas repair dynamics. Congrats to co-first authors Beeke Weinert and Stacia Wyman! And thanks to our wonderful collaborators in the Conklin labs and at AstraZeneca.

Want to give DISCOVER-seq a try? There is a very detailed protocol on protocols.io and code on Github.Feel free to reach out if you’re having trouble or want to collaborate.

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Charles moved all the way from Berkeley to Zurich with the Corn Lab. If he had stayed at Berkeley, he would have taken a qualifying exam. Things are a bit different across the pond, but there’s still a milestone at the end of the 2nd year of grad school. Charles passed with flying colors and had the honor of wearing the traditional Corn Costume.

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