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Alkhaldi, F.

Publications and source records attributed to Alkhaldi, F..

2 recordsLinked to original sources

Evolution of mechanical chromatin insulators from selfish genetic elements

Genomes require physical boundaries to separate active and repressed chromatin, a function traditionally attributed to insulator proteins. Here, we show that genomic insulation can also emerge from physical properties encoded directly within the DNA polymer. In C. elegans, transcription-coupled mutational bias remodels Helitron transposon minisatellites to match the DNA helical repeat. The resulting 10-bp periodicity (PATCs) encodes intrinsic curvature, creating topologically responsive DNA elements that favor local deformation under supercoiling, disrupt canonical nucleosome organization, and protect germline genes from progressive and heritable silencing. Rather than acting as short protein-binding motifs, Helitron-derived PATCs form extended barrier elements that limit the stabilization of repressive chromatin. Comparative analyses suggest that related sequence-encoded mechanical signatures recur across Metazoa, including at Drosophila insulators, human CTCF sites, and active human LINE-1 retrotransposons. Thus, sequence-encoded mechanics provide an evolutionarily accessible substrate for chromatin insulation--a physical layer of genome organization that can be co-opted by host genomes to protect gene expression, and potentially retained by selfish elements for their own persistence.

molecular biology↗

Modular safe-harbor Transgene Insertion (MosTI) for targeted single-copy and extrachromosomal array integration in C. elegans.

Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here we describe a modular safe harbor transgene insertion (MosTI) for use in C. elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by non-homologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25 - 75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7 - 44%) at MosTI landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for eight integrated arrays (6 to 37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of one array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

genetics↗