Biology subjects

Shimamura, A.

Publications and source records attributed to Shimamura, A..

4 recordsLinked to original sources

bioRxiv2025

Analyzing long-read CRISPR experiments with CRISPRLungo

Long-read sequencing can characterize complex genome editing-induced DNA sequence changes such as large deletions, insertions, and inversions that are difficult to detect using short-read sequencing. However, PCR amplification and sequencing errors complicate accurate variant detection, and existing analysis tools are not optimized for gene editing specific allelic outcomes. Here we present CRISPRLungo, a computational pipeline specifically designed for long-read amplicon sequencing of gene edited samples. CRISPRLungo incorporates unique molecular identifier (UMI)-based error correction and statistical filtering to distinguish true editing events from background noise, enabling robust detection of small indels and structural variants. Through systematic benchmarking using simulated datasets, we demonstrate that CRISPRLungo outperforms existing approaches in both accuracy and read recovery. CRISPRLungo supports both Oxford Nanopore and PacBio platforms and identify previously undetected structural variant edits such as inversions in published CRISPR datasets. To demonstrate allele-specific edit quantification, we applied CRISPRLungo to analyze edited primary cells from a patient with harboring compound heterozygous SBDS mutations, accurately quantifying SBDS editing outcomes despite contaminating reads from the homologous SBDSP1 pseudogene. To maximize accessibility, we developed a fully client-side web application requiring no installation, making advanced long-read analysis accessible to researchers regardless of computational expertise. CRISPRLungo is freely available at https://github.com/pinellolab/CRISPRLungo with a user-friendly web interface available at https://pinellolab.github.io/CRISPRLungo.

bioinformatics↗

bioRxiv2025

Direct and indirect regulation of fetal globin transcript by RNA-binding protein IGF2BP1

Despite extensive investigation, the molecular control of developmental hemoglobin expression remains incompletely elucidated. Hemoglobin switching is controlled by transcription factors, miRNAs, and RNA-binding proteins (RBPs) that enforce gene regulatory changes through development. Here we examine the role of the heterochronically silenced N-6 methyladenosine (m6A) RNA-binding protein IGF2BP1 that was previously described to regulate HBG1/2 indirectly by suppressing BCL11A expression through an unknown mechanism. We find that IGF2BP1 binds and activates HIC2, itself a BCL11A repressor. Furthermore, we identify that IGF2BP1 plays a BCL11A-independent role by direct binding to HBG1/2 to promote its translation. Stop codon-proximal m6A-modified coding sequences within HBG2 transcripts are necessary and sufficient for direct positive regulation mediated by IGF2BP1. This work deepens the mechanistic understanding of hemoglobin switching and suggests a physical relationship between heterochronic RBPs and globin transcripts.

genetics↗

bioRxiv2020

Distinct genetic pathways define pre-leukemic and compensatory clonal hematopoiesis in Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with predisposition to developing leukemia. We found that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. EIF6 mutations cause functional compensation for the germline deficiency by alleviating the SDS ribosome joining defect, improving translation, and reducing p53 activation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. We link development of leukemia with acquisition of biallelic TP53 alterations. Our results define distinct pathways of clonal selection driven by germline fitness constraint and provide a mechanistic framework for clinical surveillance.

cancer biology↗

bioRxiv2018

Human bone marrow disorders recapitulated in vitro using organ chip technology

Understanding human bone marrow (BM) pathophysiology in the context of myelotoxic stress induced by drugs, radiation, or genetic mutations is of critical importance in clinical medicine. However, study of these dynamic cellular responses is hampered by the inaccessibility of living BM in vivo. Here, we describe a vascularized human Bone Marrow-on-a-Chip (BM Chip) microfluidic culture device for modeling bone marrow function and disease states. The BM Chip is comprised of a fluidic channel filled with a fibrin gel in which patient-derived CD34+ cells and bone marrow-derived stromal cells (BMSCs) are co-cultured, which is separated by a porous membrane from a parallel fluidic channel lined by human vascular endothelium. When perfused with culture medium through the vascular channel, the BM Chip maintains human CD34+ cells and supports differentiation and maturation of multiple blood cell lineages over 1 month in culture. Moreover, it recapitulates human myeloerythroid injury responses to drugs and gamma radiation exposure, as well as key hematopoietic abnormalities found in patients with the genetic disorder, Shwachman-Diamond Syndrome (SDS). These data establish the BM Chip as a new human in vitro model with broad potential utility for studies of BM dysfunction.

bioengineering↗