Lab News

Eric joined the Corn lab as a Postdoc in November 2020. He has received his PhD in Biochemistry, Molecular Biology, and Biophysics from the University of Minnesota in 2020 in the lab of Dr. Wendy Gordon. He is interested in studying synthetic lethalities and viabilities in DNA repair utilizing CRISPR.

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Roman joined the Corn lab in October 2020. Earlier this year he has received his Ph.D. from the University of Boulder, where he had developed tools by combining CRISPR and aptamer components in the group of Prof. Batey. He is currently interested in finding new ways to understand DNA damage repair and developing new tools to easily modify DNA.

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Susanne joined the Corn Lab in September 2020 to support the team with High Throughput sequencing applications. She received her PhD in Paleogenomics at the University of Mainz. Susanne has a long history in Next Generation Sequencing, starting with sequencing of ancient genomes. After working in a clinical setting, she supported several research groups of the University/ETH Zurich in their genomics and transcriptomics applications using Illumina workflows. Susanne will be working at GEML implementing new protocols and sequencing strategies in genome editing workflows.

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John received his PhD in Oncology from the University of Oxford in 2020, working with Prof. Kristijan Ramadan on the molecular mechanisms of DNA-protein crosslink repair. He joined the Corn Lab as a postdoctoral researcher in October 2020. His research interests include multiplexed genome engineering, DNA repair, and synthetic lethality.

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When using CRISPR genome editing in stem cells, it’s far easier to break a gene with indels than to fix it with HDR. This manifests in an interesting way. If you monitor a “CD34+” population of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow, indels start high and stay high but HDR alleles are lost over time. Why do these different genetic outcomes differ over time? Is HDR bad for the long-term stem cells? Or is editing in the CD34+ population actually heterogeneous, and different cells get different alleles? New work from postdoc Jenny Shin in the lab, out in Cell reports, both answers this question and finds a way to fix the problem.

Jenny and collaborators used a powerful combination of immunophenotyping, next generation sequencing, and single-cell RNA-sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult human CD34+ HSPCs. These HSPCs are actually several different types of cells, including more differentiated progenitors that cycle and very “stemmy” long-term HSCs that are quiescent. The team found that there is a dramatic tension between HDR and quiescence in LT-HSCs.  Quiescent stem-enriched cells utilize NHEJ and exhibit almost no HDR. By contrast, non-quiescent cells with the same immunophenotype utilize both NHEJ and HDR. Quiescence is critical for engraftment and stem cell maintenance, so it was now clear that all cells in the CD34+ population get indels and the cycling progenitors were getting HDR alleles, but the quiescent LT-HSCs weren’t doing HDR.

Jenny then had a very creative idea. She asked if a previously reported small molecule cocktail, “XRC”, that maintains quiescence could be used after the fact to re-quiesce LT-HSCs. Using this new strategy and good timing, she found a way to get LT-HSCs with high levels of HDR by briefly allowing them to cycle during editing, and then inducing quiescence later on. This yielded a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and during long-term engraftment in mouse experiments. The re-quiescence strategy might in future be combined with engineered Cas9-geminin constructs that reduce NHEJ, further tipping the balance towards HDR. Jenny’s results highlight the tradeoffs between editing and fundamental cellular physiology and suggests strategies to manipulate quiescent cells for research and therapeutic genome editing. 

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Pia received her Bachelor’s degree in Biology from ETH Zürich in 2018. She joined the Corn Lab as a Master student in February 2020 working on a CRISPR-based screen for regulators in B-cell leukemia. Her research interests include genome engineering, functional genomics, cancer, and DNA repair mechanisms.

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Moritz joined the Corn lab as a PhD student in July 2020. He has received his Master’s degree in Molecular Biology from the University of Vienna in 2020. Moritz’ research interests include functional genomics, therapeutic gene editing and cancer biology, with a special focus on technology development.

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Sebastian joined the Corn Lab as a Master student in September 2019 after having received his Bachelor degree in Biological Sciences from the University of Konstanz in 2018. He is investigating Fanconi Anemia and the therapeutic potential of CRISPR based systems for this genetic disease.

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When my lab published editing of the sickle beta globin gene, together with Mark Walters and David Martin, we noticed something quite unexpected. Every time we targeted beta globin, we saw fetal globin go up! We also noticed this in beta globin editing papers from the labs of Paula Cannon and Matt Porteus. Transient stress can cause blood cells to express fetal globin, so at first we thought that we were just seeing something short-lived. But it turned out that fetal globin persisted after long-term transplantation of edited human cells into a mouse model. What could be happening in these cells? Could this be a route to treat globin-related diseases such as sickle cell disease? If so, why is beta thalassemia (lack of beta globin) not automatically compensated by fetal globin expression? A new paper from PhD student Mandy Boontanrart, out now in Cell Reports, finally figures out what’s going on. This work was completely driven by Mandy, using a wide range of techniques to dig into a difficult problem.

Editing itself can be stressful to cells, but Mandy’s first big findings were that the fetal globin response is not related to the the process of editing. Doing a scan of CRISPR cutting guides through beta globin, she found that any guide targeting an exon causes fetal globin to go up. But equally effective guides that target introns have no effect. She also found that CRISPRi transcriptional repression to knockdown beta globin does increase fetal globin. Therefore, it was something special about lacking beta globin, which is known as B0-stress.

Using clones of blood precursors edited to lack beta globin, Mandy did RNA-seq during differentiation to figure out how cells responded over time. The number one pathway by far was ATF4 signalling. But in a very strange way! It turned out that ATF4 signalling actually went down in cells with beta globin knockout. This was surprising because ATF4 responds to unfolded proteins, it had been suggested that lack of beta globin would lead to alpha chain aggregation that should turn on the unfolded protein response. We were seeing the exact opposite!

This actually makes sense in the context of recent work from the lab of Gerd Blobel, who found out that knockout of the heme-responsive kinase HRI can also increase fetal globin. HRI is upstream of eIF2a and ATF4, so it seemed that free heme (caused by lack of complete adult hemoglobin tetramers) had a stronger role than the free alpha chains in our system.

But what was ATF4 doing to increase fetal globin? Mandy investigated this using a large number of isogenically-controlled ChIP-seq experiments. She found that ATF4 doesn’t bind anywhere in the globin locus, nor does it bind anywhere near BCL11A, which is one of the latest hot globin regulators. In fact, Mandy found that ATF4’s effect on fetal globin still happened in K562 cells, which don’t express any BCL11A at all. Instead, Mandy found strong evidence that ATF4 regulates Myb through binding to a known enhancer region. Myb is itself a regulator of Bcl11A, but can also regulate fetal globin through other factors such as KLF1.

As always there’s a lot more to figure out. And it’s still unclear if we can use these findings to make a difference for patients with globinopathies. But thanks to Mandy’s work, we finally know why fetal globin increases when beta globin is reduced. We also have some clues as to why beta thalassemia is not always compensated by increased fetal globin. In fact, to some extent, it is! Many people with beta thalassemia exhibit increased fetal globin, but not enough to alleviate the symptoms of complete lack of beta globin. ATF4 does many jobs, so it could be that reducing ATF4 enough to yield large amounts of fetal globin might be bad for other biologies. This also adds a note of caution to targeting HRI as a therapeutic angle to increase fetal globin, since doing so could have pleiotropic effects. But all of that remains to be tested!

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