Biology subjects

Mai, M.

Publications and source records attributed to Mai, M..

5 recordsLinked to original sources

bioRxiv2025

Preventing large deletions and chromosome loss in engineered human primary T cells by CasPlus with optimized guide RNAs

Genetically engineered T-cell therapies rely heavily on genome editing tools, such as the CRISPR/Cas9 system. However, unintended on-target chromosomal alterations, including large deletions and chromosome loss can occur and pose significant risks including tumorigenesis. Here we combined CasPlus and optimized guide RNAs to reduce these issues in CRISPR/Cas9 engineering human primary T cells. CasPlus, which integrates an engineered T4 DNA polymerase with Cas9 nuclease and guide RNA, promotes favorable small insertions (1-2 bp) while reducing large deletions and chromosome loss in T cells. Our optimized guide RNAs favoring small insertions reduced large deletions and chromosome loss by two- to five-fold versus those favoring small deletions. Moreover, combining optimized guide RNA with T4 DNA polymerase further synergistically reduced large deletions and chromosome loss by additional two-fold. Notably, replacing currently used guide RNA pairs in clinically applications with optimized pairs biased towards small insertions, along with CasPlus instead of Cas9, for editing greatly reduced large deletions and chromosome loss in gene-edited human primary T cells. These findings demonstrated that pre-selecting target sites favoring small insertions via guide RNA optimization coupled with CasPlus editing is a safer and more effective strategy to improve genome stability in T-cell engineering and other gene-editing applications.

bioengineering↗

bioRxiv2024

Sulfite oxidase deficiency causes persulfidation loss and H2S release

Sulfite oxidase (SOX) deficiency is a rare inborn error of cysteine metabolism resulting in severe neurological damage. In patients, sulfite accumulates to toxic levels causing a raise in downstream products S-sulfocysteine (SSC), mediating excitotoxicity, and thiosulfate, a catabolic intermediate/product of H2S metabolism. Here, we report a full-body knock-out mouse model for SOX deficiency (SOXD) with a severely impaired phenotype. Amongst the urinary biomarkers, thiosulfate showed a 45-fold accumulation in SOXD mice representing the major excreted S-metabolite. Consistently, we found increased plasma H2S, which was derived from sulfite-induced release from persulfides as demonstrated in vitro and in vivo. Mass spectrometric analysis of total protein persulfidome identified a major loss of persulfidation in 20% of the proteome affecting enzymes in amino acids and fatty acid metabolism. Urinary amino acid profiles indicate metabolic rewiring suggesting partial reversal of the TCA cycle thus identifying a novel contribution of H2S metabolism and persulfidation in SOXD.

biochemistry↗

bioRxiv2024

In vitro neutrophil-bacteria assay in whole blood microenvironments with single-cell confinement

Blood is a common medium through which invasive bacterial infections disseminate in the human body. In vitro neutrophil-bacteria assays allow flexible mechanistic studies and screening of interventional strategies. In standard neutrophil-bacteria assays, both the immune cells and microorganisms are typically interrogated in an exogenous, homogeneous, bulk fluid environment (e.g., culture media or bacterial broth in microtiter plates), lacking the relevant physicochemical factors in the heterogenous blood-tissue microenvironment (e.g., capillary bed) with single-cell confinement. Here we present an in vitro neutrophil-bacteria assay by leveraging an open microfluidic model known as "-Blood" that supports sub-microliter liquid microchannels with single-cell confinement. In this study we compare the exogenous and endogenous fluids including neutrophils in RPMI (standard suspension cell culture media) and whole blood in response to Staphylococcus aureus (S. aureus, a gram-positive, non-motile bacterium) in phosphate buffered saline (PBS), Mueller Hinton Broth (MHB), and human serum. Our results reveal a significant disparity between the exogenous and endogenous fluid microenvironments in the growth kinetics of bacteria, the spontaneous generation of capillary (i.e., Marangoni) flow, and the outcome of neutrophil intervention on the spreading bacteria.

biophysics↗

bioRxiv2023

Identification of residues potentially involved in optical shifts in the water-soluble chlorophyll-a binding protein through molecular dynamics simulations

Reversible light- and thermally-induced spectral shifts are universally observed in a wide variety of pigment-protein complexes, at temperatures ranging from cryogenic to ambient. They can be observed either directly, in single-molecule spectroscopy experiments, or via non-photochemical spectral hole burning. These shifts are important to understand, for example, to gain a clearer picture of the primary processes of photosynthesis, or of general features of the protein energy landscapes. In this article, we have employed large-scale molecular dynamics simulations of a prototypical pigment-protein complex to better understand these shifts at a molecular scale. Although multiple mechanisms have been proposed over the years, no verification of these proposals via MD simulations has thus far been performed; our work represents the first step in this direction. The common requirement for all these mechanisms is the presence of doublewell (or multiple-well) features of the protein energy landscapes. In this work, from large-scale molecular dynamics simulations of the Water-Soluble Chlorophyll-binding Protein complex, we identified side chain rotations of certain amino acid residues as likely candidates for relevant multi-well landscape features. The protein free energy landscapes associated with side chain rotations feature energy barriers of around 1100- 1600 cm-1, in agreement with optical spectroscopy results, with the most promising residue type associated with experimental signatures being serine, which possesses a symmetric landscape and moment of inertia of a relevant magnitude.

biophysics↗

bioRxiv2023

Phage DNA polymerase prevents on-target damage and enhances precision of CRISPR editing

Common unintended chromosomal alterations induced by CRISPR/Cas9 in mammalian cells, particularly on-target large deletions and chromosomal translocations present a safety challenge for genome editing. Base editing and prime editing that can precisely introduce desired edits without double-stranded breaks and exogenous DNA templates face their own challenges. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified T4 DNA polymerase derived from phage T4 that greatly prevents undesired on-target large deletions and chromosomal translocations while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency to correct common frameshift mutations in DMD (exon 52 deletion) and restored higher level of dystrophin expression than Cas9-alone in human induced pluripotent stem cell-derived cardiomyocytes. Moreover, CasPlus can greatly reduce the frequency of on-target large deletions in mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus represses chromosomal translocations while maintaining gene disruption efficiency that is higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

bioengineering↗