Biology subjects

Konishi, C. T.

Publications and source records attributed to Konishi, C. T..

2 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

Modeling and Correction of Protein Conformational Disease in iPSC-derived Neurons through Personalized Base Editing

AbstractAltered protein conformation can cause incurable neurodegenerative disorders. Mutations in SERPINI1, the gene encoding neuroserpin, can alter protein conformation resulting in cytotoxic aggregation leading to neuronal death. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant progressive myoclonic epilepsy that progresses to dementia and premature death. We developed HEK293T and induced pluripotent stem cell (iPSC) models of FENIB, harboring a patient-specific pathogenic SERPINI1 variant or stably overexpressing mutant neuroserpin fused to GFP (MUT NS-GFP). Here, we utilized a personalized adenine base editor (ABE)-mediated approach to correct the pathogenic variant efficiently and precisely to restore neuronal dendritic morphology. ABE-treated MUT NS-GFP cells demonstrated reduced inclusion size and number. Using an inducible MUT NS-GFP neuron system, we identified early prevention of toxic protein expression allowed aggregate clearance, while late prevention halted further aggregation. To address several challenges for clinical applications of gene correction, we developed a neuron-specific engineered virus-like particle to optimize neuronal ABE delivery, resulting in higher correction efficiency. Our findings provide a targeted strategy which may treat FENIB and potentially other neurodegenerative diseases due to altered protein conformation such as Alzheimers and Huntingtons diseases.

molecular biology↗