Lab News

Increasing HDR by timed inhibition of CDC7, published in Nature Communications

When doing genome editing, fixing sequences by HDR is better than breaking them by making indels. If you really want to break something, you could even use HDR...

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When doing genome editing, fixing sequences by HDR is better than breaking them by making indels. If you really want to break something, you could even use HDR to insert a precise indel or a stop codon. Unfortunately, HDR is relatively inefficient in human cells. Single stranded oligo donors help, but editing the same locus with a double stranded plasmid DNA donor is almost always painful. What’s the bottleneck? We tried to answer this question in a new paper just out in Nature Communications.

To answer this question, Chris Richardson and Beeke Wienert led a superstar team to perform CRISPRi screening while simultaneously editing with a double stranded DNA plasmid donor. The screen itself was performed by Sharon Feng, a superstar undergraduate. Putting everything together was an exciting collaboration with the labs of Bruce Conklin and Alex Marson.

The first set of hits are known homologous recombination factors, such as BRCA1. This gives high confidence that the screen worked as expected. Surprisingly, the same Fanconi Anemia complexes that are required for single stranded oligo HDR are required for plasmid HDR. The FA pathway is thus a core regulator of all forms of HDR!

But we really wanted to know genes could increase HDR if they were removed. Knocking down a gene is hard to do in many contexts. So we focused on genes with known inhibitors. It turns out that small molecule inhibitors of CDC7 give very nice boosts in HDR from both single stranded oligo and plasmid DNA donors. This works for small changes (SNPs), medium changes (adding epitope tags) and even large cargoes (site-targeted transgenes). It also works in a variety of cell types, including hematopoietic stem cells and T cells. Not every cell is created equally, so check out the paper for detailed guidelines.

Our favorite CDC7 inhibitor is XL413, which is non-toxic and quite reversible. This distinguishes it from some other HDR-improving compounds that lead big genomic messes, including polyploidy. Delving into mechanism, CDC7 inhibition leads to loss of MCM2 phosphorylation. Because MCM2 phosphorylation is required for S phase progression, XL413 leads to a longer S phase. This is a magical phase of the cell cycle for HDR, and so our model is that XL413 increases HDR by increasing the amount of time cells are able to do HDR. We tested this with a timing experiment. Hitting cells with Cas9 and then immediately putting them in XL413 leads to increased HDR, because the cells are piling up in S phase at the same time they are repairing the Cas9 damage. But putting cells in XL413 first and then taking them out during editing leads to decreased HDR. This is because the cells all pile up S phase before editing, and then exit into HDR non-permissive cell cycle while Cas9 is doing its thing.

We hope other labs find XL413 to be useful to increase HDR. It’s not a magic bullet and seems to work especially well in hematopoietic lineages and iPSCs. If you try it out in your favorite cells, please let us know your experience!

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Welcome to Lena

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...

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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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ER-autophagy screen published in Cell

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...

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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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Choosing the right path in genome editing – review published in Nature Cell Biology

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 ...

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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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CDN transporter collaboration with David Raulet published in Nature

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....

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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

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...

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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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MCL1/CDK9 screen published in eLife

Our CRISPR screening projects are starting to come out! Today work from postdoc Shaheen Kabir, in collaboration with oncologists at AstraZeneca, was published in eLife. Shaheen used a very creative FACS screen to find genes involved in the early apoptotic response to CDK9 and MCL1 inhibitors. Inhibition of CDK9 reduces transcript half-life and indirectly inhibits MCL1, whereas the other compound screen directly binds MCL1. Several cancers respond well to these new compounds, but others are already completely resistant. Shaheen went looking for genes involved in this resistance and found some very interesting shared hits. She focused her mechanistic work on the CUL5 ubiquitin ligase complex, which is a multi-component system used to degrade target proteins. Almost every component of the CUL5 complex was a hit in the screen, and Shaheen found that knockdown of CUL5 components affected the stability of pro-apoptotic proteins Bim and Noxa. CUL3 type ligases are already established cancer targets, and Shaheen’s work shows that CUL5 could also be targeted to synergize with front-line cancer therapeutics. Congratulations Shaheen!

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Welcome Erman, Kinga, and Markus

From left to right: Markus, Kinga, and Erman

Three people joined the lab, all in one day! Erman is a postdoc, interested in DNA repair and genome editing, Kinga...

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From left to right: Markus, Kinga, and Erman

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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DISCOVER-seq published in Science

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....

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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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Questions and/or comments about Corn Lab and its activities may be addressed to:

JACOB.CORN@BIOL.ETHZ.CH

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