Biology subjects

Melamed-Bessudo, C.

Publications and source records attributed to Melamed-Bessudo, C..

4 recordsLinked to original sources

bioRxiv2026

Features affecting Cas9-Induced Editing Efficiency and Patterns in Tomato: Evidence from a Large CRISPR Dataset

CRISPR/Cas9 is a cornerstone of genome editing, yet the determinants of editing efficiency and DNA Double Strand Break (DSB) repair outcomes remain poorly understood, particularly in plants. To address this gap, we generated a dataset of 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts and quantified editing efficiencies together with ATAC-seq-derived chromatin accessibility and transcriptional states in the same cellular system. Editing efficiency was consistently higher at open chromatin sites and modestly elevated in promoters and introns relative to exons, whereas transcriptional activity did not measurably influence editing outcomes. Additionally, we identified a local genomic effect resulting in less variable editing between sgRNAs targeting the same compared to different genes. A distinct subset of sgRNAs achieved nearly complete editing, producing long deletions with extended microhomology tracts. These repair footprints closely parallel those observed for high-efficiency guides in human datasets, implicating conserved sequence-driven biases and a predominant role for microhomology-mediated end joining at these sites. Yet, widely used human-trained prediction models failed to rank sgRNA performance in plants, underscoring the limits of cross-species generalization. This dataset defines how chromatin accessibility, genomic context, and intrinsic sequence characteristics shape Cas9 activity in plants, and provides a resource for improving guide design and advancing mechanistic understanding of plant DNA repair.

plant biology↗

bioRxiv2023

A CRISPR-induced DNA break can trigger crossover, chromosomal loss and chromothripsis-like rearrangements

The fate of DNA double-strand breaks (DSBs) generated by the Cas9 nuclease has been thoroughly studied. Repair via non-homologous end-joining (NHEJ) or homologous recombination (HR) is the common outcome. However, little is known about unrepaired DSBs and the type of damage they can trigger in plants. In this work, we designed a new assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (Betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay we detected a flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole genome sequencing, molecular, and cytological analyses, supports a model in which breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a new tool for precise breeding via targeted crossover detection. It also uncovers CRISPR mediated chromothripsis-lke events that had not been previously identified in plants.

genetics↗

bioRxiv2022

Machine learning reveals conserved chromatin patterns determining meiotic recombination sites in plants

Distribution of meiotic recombination events in plants has been associated with local chromatin and DNA characteristics, chromosome landmark proximity, and other features1-7. However, relative importance of these characteristics is unclear and it is unknown if they are sufficient to unambiguously determine recombination landscape8. Here, we analyzed over 40 DNA sequence, chromatin, and chromosome location features of maize and Arabidopsis recombination sites using machine learning9,10. We discovered that a combination of just three features, CG methylation, CHG methylation, and nucleosome occupancy, enabled identification of exact crossover site with 90% accuracy. These results imply redundancy of most recombination site characteristics. Recombination takes place in a small fraction of the genome with chromatin features distinct from those of genome at large. Surprisingly, crossover sites show elevated heterochromatin histone marks despite low DNA methylation. Crossover site features show broad evolutionary conservation, which will enable creating genetic maps in species where conventional mapping is unfeasible.

plant biology↗

bioRxiv2017

T-DNA integration is rapid and influenced by the chromatin state of the host genome.

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of integration showed that selected events reported on previously were associated with extremely low methylation and nucleosome occupancy. Conversely, non-selected events from this study showed chromatin marks, such as high nucleosome occupancy and high H3K27me3 that correspond to 3D-interacting heterochromatin islands embedded within euchromatin. Such structures might play a role in capturing and silencing invading T-DNA.

plant biology↗