Biology subjects

Walters, M. C.

Publications and source records attributed to Walters, M. C..

4 recordsLinked to original sources

bioRxiv2026

Combinatorial base editing couples disease correction with lineage amplification in hematopoietic stem and progenitor cells

First-generation genome editing therapies have largely focused on correcting or compensating for pathogenic variants. However, as these approaches enter the clinic, emerging biological constraints limit maximal therapeutic impact. Because globin genes are activated late during erythroid differentiation, genome-corrected hematopoietic stem and progenitor cells (HSPCs) gain little selective advantage in the bone marrow. Here, we establish a strategy that links therapeutic genome edits to an erythroid fitness-enhancing allele to amplify the output of clinically relevant cells. We develop a multiplex base editing strategy that couples fetal hemoglobin (HbF) reactivation with erythroid lineage expansion. Introduction of a naturally occurring erythropoietin receptor truncation (tEPOR) associated with benign erythrocytosis increased erythroid cell production without impairing viability or differentiation. Combinatorial editing of tEPOR together with the BCL11A erythroid enhancer and HBG1/2 promoters in healthy donor, sickle cell disease, and {beta}-thalassemia HSPCs synergistically increased erythroid proliferation and HbF expression beyond single base-edited or Casgevy-treated controls. Multiplex base-edited HSPCs retained long-term lineage repopulation and engraftment capacity in vivo, establishing a modular strategy that pairs disease correction with lineage amplification to improve therapeutic potency.

cell biology↗

bioRxiv2025

Synthesis errors in oligonucleotides are propagated into the genome by homology-directed repair

Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. By deep sequencing ssODNs from three manufacturers, and amplicons from edited hematopoietic stem/progenitor cells, we find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign {beta}-globin variants, while the less frequent frameshift deletions will generate {beta}-thalassemia alleles. Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs.

genomics↗

bioRxiv2018

In vivo selection for corrected β-globin alleles after CRISPR/Cas9 editing in human sickle hematopoietic stem cells enhances therapeutic potential

Sickle Cell Disease (SCD), one of the worlds most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that in vivo erythroid differentiation markedly enriches for corrected {beta}-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy.\n\nONE SENTENCE SUMMARYCas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

genetics↗

bioRxiv2018

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

Sickle Cell Disease and {beta}-thalassemia, which are caused by defective or deficient adult {beta}-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Expression of the fetal {beta}-like globin, also known as {gamma}-globin, can ameliorate both disorders by serving in place of the adult {beta}-globin. Here we use CRISPR-Cas9 gene editing to explore a putative {gamma}-globin silencer region identified by comparison of naturally-occurring deletion mutations associated with up-regulated {gamma}-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of fetal hemoglobin (HbF) or {gamma}-globin. Screening of individual sgRNAs in one sub-region revealed three single guides that caused mild increases in {gamma}-globin expression. However, clonal cell lines with the 1.7 kb region deleted did not up-regulate {gamma}-globin and neither did lines with either of two of sub-regions identified in the screen deleted. These data suggest that the region is not an autonomous {gamma}-globin silencer, and thus by itself is not a suitable therapeutic target in the {beta}-hemoglobinopathies.

genetics↗