Biology subjects

Corn, J.

Publications and source records attributed to Corn, J..

3 recordsLinked to original sources

bioRxiv2018

Controlled cycling and quiescence enables homology directed repair in engraftment-enriched adult hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSCs) are the source of all blood components, and genetic defects in these cells are causative of disorders ranging from severe combined immunodeficiency to sickle cell disease. However, genome editing of long-term repopulating HSCs to correct mutated alleles has been challenging. HSCs have the ability to either be quiescent or cycle, with the former linked to stemness and the latter involved in differentiation. Here we investigate the link between cell cycle status and genome editing outcomes at the causative codon for sickle cell disease in adult human CD34+ hematopoietic stem and progenitor cells (HSPCs). We show that quiescent HSPCs that are immunophenotypically enriched for engrafting stem cells predominantly repair Cas9-induced double strand breaks (DSBs) through an error-prone non-homologous end-joining (NHEJ) pathway and exhibit almost no homology directed repair (HDR). By contrast, non-quiescent cycling stem-enriched cells repair Cas9 DSBs through both error-prone NHEJ and fidelitous HDR. Pre-treating bulk CD34+ HSPCs with a combination of mTOR and GSK-3 inhibitors to induce quiescence results in complete loss of HDR in all cell subtypes. We used these compounds, which were initially developed to maintain HSCs in culture, to create a new strategy for editing adult human HSCs. CD34+ HSPCs are edited, allowed to briefly cycle to accumulate HDR alleles, and then placed back in quiescence to maintain stemness, resulting in 6-fold increase in HDR/NHEJ ratio in quiescent, stem-enriched cells. Our results reveal the fundamental tension between quiescence and editing in human HSPCs and suggests strategies to manipulate HSCs during therapeutic genome editing.

cell biology↗

bioRxiv2018

Atlastins mediate selective autophagy of the endoplasmic reticulum

The selective lysosomal degradation (autophagy) of entire organelles is required for cellular homeostasis, and its dysregulation is involved in degenerative disorders such as Parkinsons Disease. While autophagy of mitochondria (mitophagy) is becoming better understood, other forms of organelle autophagy are relatively unexplored. Here we develope multiple quantitative assays to measure organelle autophagy using flow cytometry, microscopy, and Western blotting. Focusing on autophagy of the endoplasmic reticulum (ER-phagy), we show that these assays allow facile measurement of ER-phagy, and that ER-phagy is inhibited by knockdown of either core autophagy components or the recently reported FAM134B ER-phagy receptor. Using these assays, we further identify that Atlastins, the ER-resident GTPases involved in ER membrane morphology, are key positive effectors of ER-phagy. Atlastin-depleted cells have decreased ER-phagy under starvation conditions, and Atlastins role in ER-phagy requires both a functional GTPase domain and proper ER localization. The three Atlastin family members functionally compensate for one another during ER-phagy and may form heteromeric complexes with one another. We also find that Atlastins act downstream of the FAM134B ER-phagy receptor. We propose that during ER-phagy, Atlastins remodel ER membrane to separate pieces of FAM134B-marked ER for efficient autophagosomal engulfment. Human mutations in Atlastins led to hereditary spastic paraplegia, and our results suggest that this disease may be linked to deficiencies in ER-phagy rather than ER morphology.

cell biology↗

bioRxiv2017

CRISPR-Cas9 Genome Editing In Human Cells Works Via The Fanconi Anemia Pathway

CRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

cell biology↗