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Erythrocytes, or red blood cells, carry hemoglobin and circulate throughout the body to supply oxygen. β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. These conditions result in anemia and organ damage, and available treatment options are limited. Stem cell transplantation is currently the only curative approach, although its feasibility relies on the availability of a suitable donor.

Hemoglobin is a tetrameric protein composed of 2 α-like (HBA) and 2 β-like subunits (HBB). Hemoglobin A1 (HbA1) constitutes 97% of adult hemoglobin, while Hemoglobin A2 (HbA2) makes up 2-3%. HbA2 is composed of two α-globin subunits and two δ-globin (HBD) subunits. HBD is a homologous to HBB gene, but with much lower expression compared to HBB due to a weak promoter.  Currently, many efforts are focused on increasing fetal hemoglobin (HbF) to treat the β-hemoglobinopathies. But HbA2 is more similar to HbA1 and is already expressed at low levels in all adult red blood cells. What if we were to increase HbA2 levels? Could they potentially compensate for beta-globin deficiency? Can genome editing technologies be used to boost transcriptional activity of the endogenous HBD promoter to increase HbA2 levels? Mandy Boontanrart, a Postdoc in our lab, was eager to discover the answers to these questions.

Using CRISPR-Cas9 genome editing, we inserted various transcription factor binding sequences into the endogenous HBD promoter. Team efforts yielded positive results as we successfully increased the transcriptional activity in HUDEP-2 immortalized erythroid progenitor cells, resulting in a significant upregulation of HBD expression. Despite roughly equal homology-directed repair rates between all promoter designs, we observed a significant increase in HBD only for the design with all three elements (KLF1, β-DRF, and TFIIB). We next explored whether endogenous editing of the HBD promoter can be accomplished in bone marrow stem cells. We found up to 46% HBD expression in clonal populations. We also tested a small molecule drug that enhances HDR outcomes by inhibiting the NHEJ pathway and observed an increase in the percent of HDR alleles in pooled edited bone marrow stem cells.

While our findings provide key mechanistic insight into the globin gene regulation, several questions remain to be tackled.  Is heterozygous knock-in of the promoter design in β-hemoglobinopathy cells is sufficient to ameliorate disease phenotypes? What is the safety profile of this strategy?

Overall, our work is a promising approach for restoring hemoglobin levels in red blood cells. This strategy might open new therapeutic avenues for to treating beta-hemoglobinopathies in the future.

For more, check out our paper, it is now out in Elife!

Note: Excitingly, Mandy is now leading an ETH spin-off, building upon the findings of the paper, check out their brand-new website https://www.ariyabio.ch/!

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The Coronavirus lockdowns this spring disrupted many projects and students. But when life gives you lemons, make lemonade. In our lab, almost everyone took the stay-at-home orders as motivation to learn some coding. Sebastian Siegner, a Masters’ student at the time and now recently joined for a full Ph.D., had been working on proof-of-concept experiments for therapeutic base editing and prime editing. But he was frustrated with designing base editor gRNAs and newly-described prime editor pegRNAs by hand. So he used the lockdown to write PnB Designer, which is a fast and scalable helper to design your prime and base editing experiments. Check out the paper in BMC Bioinformatics.

PnB designer can be used to design base editing gRNAs and prime editing pegRNAs in both single-edit and batch mode. You can design against arbitrary DNA sequences (copy/paste your sequence as input), or you can enter genomic coordinates of your favorite gene in your favorite genome. Several species are currently supported, from human to plant. For base editors, just choose which nucleotide change you want to make and the software will take into account both the mutation and editing window to figure out the best editor to use. For prime editing, you can test all kinds of reverse transcriptase template (RTT) and primer binding site (PBS) lengths with a click of a button. The output is a table of possible gRNAs/pegRNAs, ordered by a heuristic score.

Sebastian tested PnB Designer by designing pegRNAs to model most of the human disease-associated mutations in ClinVar using prime editing. He even varied RTT and PBS length for each of these 96,000+ mutations, figuring out good parameters to keep pegRNAs at a reasonable length while still modelling ~80% of all variants.

This was a challenging but exciting side-project for Sebastian’s Masters’ degree. He wrote PnB Designer independently during the lockdown, and the rest of the lab acted as beta testers to provide suggestions. Congrats to Sebastian on your first paper, which is already being used by several people in the Zurich area. PnB Designer is completely free to use and is hosted by the Functional Genomics Center Zurich.

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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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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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Are you interested in working on genome editing, DNA repair, and organelle quality control in a dynamic lab environment? We are seeking a technician/lab manager and a bioinformatics scientist. Apply at the links below.

Technician / Lab Manager

Bioinformatics Scientist

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We had an slightly belated American Thanksgiving in Zürich, complete with turkey, stuffing, mashed potatoes, and gravy. After eating more than our fill, we all walked down to a big Weinachtsmarkt for Glühwein. A great start to the holiday season!

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