Biology subjects

Abebe, J. S.

Publications and source records attributed to Abebe, J. S..

3 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

Nanopore Guided Annotation of Transcriptome Architectures

High-resolution annotations of transcriptomes from all domains of life are essential for many sequencing-based RNA analyses, including Nanopore direct RNA sequencing (DRS), which would otherwise be hindered by misalignments and other analysis artefacts. DRS allows the capture and full-length sequencing of native RNAs, without recoding or amplification bias, and resulting data may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails on individual RNA molecules. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene dense organisms such as viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS datasets and developed a novel approach to transcriptome. We demonstrate, using a combination of synthetic and original datasets, that our novel approach yields a high level of precision and recall when reconstructing both gene sparse and gene dense transcriptomes from DRS datasets. We further apply this approach to generate a new high resolution transcriptome annotation of the neglected pathogen human adenovirus type F 41 for which we identify 77 distinct transcripts encoding at least 23 different proteins.

bioinformatics↗

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↗