Biology subjects

Biondi, T.

Publications and source records attributed to Biondi, T..

4 recordsLinked to original sources

bioRxiv2024

Engineered Bxb1 variants improve integrase activity and fidelity

Many current genome editing technologies rely on the action of large serine integrases (LSIs) to insert gene-sized DNA sequences into the genome. Bxb1 is the most commonly used LSI for therapeutic efforts, including PASTE, PASSIGE and I-PGI. While Bxb1 demonstrated good activity in vitro in cycling cells, the activity in non-dividing hepatocytes was significantly less efficient. Further, wild-type Bxb1 is known to have detectable off-target activity at cryptic genomic sites, which presents a potential safety risk for therapeutic development. To address these issues, we developed Bxb1 variants that demonstrate increased specificity and potency in vitro and have engineered stabilized Bxb1 variants that increase in vivo activity over 25-fold enabling targeted integration at therapeutically relevant levels.

biochemistry↗

bioRxiv2024

Large Serine Integrase Off-Target Discovery with Deep Learning for Genome Wide Prediction

Large Serine Integrases (LSIs) hold significant therapeutic promise due to their ability to efficiently incorporate gene-sized DNA into the human genome, offering a method to integrate healthy genes in patients with monogenic disorders or to insert gene circuits for the development of advanced cell therapies. To advance the application of LSIs for human therapeutic applications, new technologies and analytical methods for predicting and characterizing off-target recombination by LSIs are required. It is not experimentally tractable to validate off-target editing at all potential off-target sites in therapeutically relevant cell types because of sample limitations and genetic variation in the human population. To address this gap, we constructed a deep learning model named IntQuery that can predict LSI activity genome-wide. For Bxb1 integrase, IntQuery was trained on quantitative off-target data from 410,776 cryptic attB sequences discovered by Cryptic-seq, an unbiased in vitro discovery technology for LSI off-target recombination. We show that IntQuery can accurately predict in vitro LSI activity, providing a tool for in silico off-target prediction of large serine integrases to advance therapeutic applications.

bioinformatics↗

bioRxiv2024

Large Serine Integrase Off-target Discovery and Validation for Therapeutic Genome Editing

While numerous technologies for the characterization of potential off-target editing by CRISPR/Cas9 have been described, the development of new technologies and analytical methods for off-target recombination by Large Serine Integrases (LSIs) are required to advance the application of LSIs for therapeutic gene integration. Here we describe a suite of off-target recombination discovery technologies and a hybrid capture validation approach as a comprehensive framework for off-target characterization of LSIs. HIDE- Seq (High-throughput Integrase-mediated DNA Event Sequencing) is a PCR-free unbiased genome-wide biochemical assay capable of discovering sites with LSI- mediated free DNA ends (FDEs) and off-target recombination events. Cryptic-Seq is a PCR-based unbiased genome-wide biochemical or cellular-based assay that is more sensitive than HIDE-Seq but is limited to the discovery of sites with off-target recombination. HIDE-Seq and Cryptic-Seq discovered 38 and 44,311 potential off-target sites respectively. 2,455 sites were prioritized for validation by hybrid capture NGS in LSI- edited K562 cells and off-target integration was detected at 52 of the sites. We benchmarked the sensitivity of our LSI off-target characterization framework against unbiased whole genome sequencing (WGS) on LSI-edited samples, and off-target integration was detected at 5 sites with an average genome coverage of 40x. This reflects a greater than 10-fold increase in sensitivity for off-target detection compared to WGS, however only 4 of the 5 sites detected by WGS were also validated by hybrid capture NGS. The dissemination of these technologies will help advance the application of LSIs in therapeutic genome editing by establishing methods and benchmarks for the sensitivity of off-target detection.

genomics↗

bioRxiv2022

The telomere-to-telomere, gapless, phased diploid genome and methylome of the green alga Scenedesmus obliquus UTEX 3031 reveals significant heterozygosity and functional separation of the haplotypes

Recent advances in sequencing technologies have improved contiguity of de novo genome assemblies. Nevertheless, the genomes of all eukaryotic organisms which are polyploid remain unfinished, limiting understanding of genetic and structural variation in diploid or polyploid organisms. Herein, we report the methodology and analysis of a 100% complete, gapless, phased, telomere-to-telomere diploid genome assembly of the eukaryote, Scenedesmus obliquus UTEX 3031 (DOE0152Z). Analysis of the fully assembled and resolved haplotypes revealed significant genomic rearrangements. Inter-haplotype heterogeneity was significant on most chromosomes yet one chromosome pair (Chromosome 15) was found to contain nearly no heterozygosity. Analysis of the 5mC methylation patterns revealed divergence in active gene content across haplotypes. Assembly of fully resolved chromosome pairs enabled complete resolution of genomic rearrangements and heterogeneity of haplotypes, the genomic basis of trait gain/loss, and evolutionary divergence across chromosome pairs. Further, when combined with 5mC methylation patterns, the assembly provides critical annotation information for genetic engineering approaches to achieve full knock-outs in allelic pairs.

genomics↗