Biology subjects

Norouzi, D.

Publications and source records attributed to Norouzi, D..

7 recordsLinked to original sources

bioRxiv2025

Highly Efficient Multiplexed Genome Engineering and Clone Selection to Enable Next Generation Induced Pluripotent Stem Cell (iPSC)-based Cell Therapies

Human induced pluripotent stem cells (iPSCs) have revolutionized regenerative medicine and cellular therapies. To improve the functionality and safety of iPSC-based therapies, genome engineering has been employed to disrupt gene expression and introduce therapeutic transgenes. However, current genome editing methods face significant challenges, including labor-intensive procedures, low efficiency, and safety concerns. Here, we report a novel and efficient approach for multiplex genome engineering in iPSCs using Integrase-mediated Programmable Genomic Integration (I-PGI) technology. I-PGI combines CRISPR-mediated genome editing and site-specific integrases, enabling precise gene insertion without DNA double-strand breaks. By optimizing I-PGI components and protocols, we achieved multiple gene knockouts and knock-ins of up to five transgenes in a single process. Additionally, we developed a streamlined workflow for enrichment, deposition, and screening of single-cell clones with the desired genome edits. This approach significantly accelerates and improves the precision of multiplex iPSC engineering, paving the way for next-generation iPSC-based therapies.

genomics↗

bioRxiv2025

A Super-Loop Extrusion Mechanism Shapes the 3D Mitotic Chromosome Folding

Despite significant advances in understanding mitotic chromosome folding, existing loop-extrusion models - based on two nested tiers of Condensin I- and II-mediated loops - fail to fully reproduce the structural features of chromosomes observed in vivo. We propose a hierarchical three-layer model in which a third set of megabase-scale "super-loops," extruded by a small number of high-processivity motors, compacts chromatin to its native dimensions. Polymer simulations of human chromosome 1 (250 Mb) show that 25-200 consecutive or interlaced super-extruders forming [~]10 Mb loops are sufficient to collapse a 25 m bottle-brush structure into the empirically observed 5-8 x 1.5 m rod. This model simultaneously reproduces the second diagonal in mitotic Hi-C maps, the double-helical scaffold seen in 3D-SIM microscopy, and the alternating-handedness, zero-mean helicity reported by high-resolution imaging. Interlaced super-loops enhance structural robustness against extruder loss and naturally account for the centromere as a region devoid of super-extruders. Although our simulations remain agnostic to molecular identity, we hypothesize that specialized forms of Condensin II, aided by auxiliary chromosome-associated proteins, act as super-extruders, while Topoisomerase II and other cross-linkers stabilize accumulated stress by converting transient DNA crossings into durable catenations. This three-layered mechanism resolves longstanding discrepancies in chromosome size, shape, contact-map profile, and scaffold ultrastructure within a unified physics-based framework. The next step is to identify or distinguish super-extruders experimentally and to design Hi-C, imaging, or mechanical perturbation assays to validate their existence and dynamics.

genomics↗

bioRxiv2024

Ligase-mediated programmable genomic integration (L-PGI): an efficient site-specific gene editing system that overcomes the limitations of reverse transcriptase-based editing systems

Since their discovery, CRISPR/Cas9 systems have been repurposed for programmable targeted genomic editing. This has led to unprecedented advancement of gene editing for therapeutic benefit. Initial uses of CRISPR/Cas9 were focused on gene disruption via DNA cleavage, but significant engineering led to systems for single base editing as well as insertion, deletion and manipulation of short stretches of genomic sequences using nicking Cas9 and RT-based methods. These technologies allowed safer and more precise editing but were limited to small corrections and showed significantly reduced efficiencies in nondividing cells, presenting difficulty for translation to in vivo therapies. To find an alternate editing strategy that could address these shortcomings, we revisited the mechanism of DNA nicking by nCas9. nCas9 nicking creates a free 5 phosphate group and a 3 hydroxyl group on the complementary strand of the target sequence. Under ordinary conditions in the cell these ends are re-joined by endogenously expressed ligases to repair DNA back to wild-type. If, however, a DNA fragment containing the desired edit were present, ligation of the nicked genomic DNA with the delivered fragment could result in gene editing. We demonstrate that optimization of each component and introduction of a chemically modified high affinity splinting DNA allows a variety of ligase-based edits, including longer edits not efficient with RT-based systems, at high efficiencies and fidelities that minimize genomic byproducts in both dividing and nondividing cells as well as in vivo in adult mice. Here we present the first therapeutically relevant ligation-based programmable gene editing technology, L-PGI.

bioengineering↗

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↗

bioRxiv2020

Multi-contact 3C data reveal that the human genome is largely unentangled

The genome is organized into chromosome territories that are themselves spatially segregated in A and B compartments. The extent to which interacting compartment domains and chromosomes are topologically entangled is not known. We show that detection of series of co-occurring chromatin interactions using multi-contact 3C (MC-3C) reveals insights into the topological entanglement of compartment domains and territories. We find that series of co-occurring interactions and their order represent interaction percolation paths through nuclear space in single cells where fragment 1 interacts with fragment 2, which in turn interacts with fragment 3 and so on. Analysis of paths that cross two chromosome territories revealed very little mixing of chromatin from the two chromosomes. Similarly, paths that cross compartment domains show that loci from interacting domains do not mix. Polymer simulations show that such paths are consistent with chromosomes and compartment domains behaving as topologically closed polymers that are not catenated with one another. Simulations show that even low levels of random strand passage, e.g. through topoisomerase II activity, would result in entanglements and mixing of loci of different chromosomes and compartment domains with concomitant changes in interaction paths inconsistent with MC-3C data. Our results show that cells maintain a largely unentangled state of chromosomes and compartment domains.

genomics↗

bioRxiv2018

Polymorphic 30-nm Chromatin Fiber and Linking Number Paradox. Evidence for the Occurrence of Two Distinct Topoisomers.

We discuss various models of spatial organization of the 30-nm fiber that remains enigmatic despite 40 years of intensive studies. Using computer simulations, we found two topologically different families of the fiber conformations distinguished by the linker length, L: the fibers with L = {10n} and {10n+5} bp have DNA linking numbers per nucleosome DLk >> -1.5 and -1.0, respectively. The fibers with DLk >> -1.5 were observed earlier, while the topoisomer with DLk >> -1.0 is novel. These predictions were confirmed for circular nucleosome arrays with precisely positioned nucleosomes. We suggest that topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. In particular, the {10n+5} DNA linkers are likely to produce transcriptionally competent chromatin structures. This hypothesis is consistent with available data for several eukaryotes, from yeast to human. We also analyzed two recent studies of chromatin fibers - on the nucleosome crosslinking in vitro, and on radioprobing DNA folding in human cells. In both cases, we show that the novel topoisomer with DLk >> -1.0 has to be taken into account to interpret experimental data. This is yet another evidence for occurrence of two distinct fiber topoisomers. Potentially, our findings may reflect a general tendency of chromosomal domains with different levels of transcription to retain topologically different higher-order structures.

molecular biology↗

bioRxiv2017

DNA topology in chromatin is defined by nucleosome spacing

In eukaryotic chromatin, DNA makes about 1.7 left superhelical turns around an octamer of core histones implying that formation of nucleosomes would alter the overall topology of DNA by a comparable difference of the DNA linking number ({Delta}Lk) per nucleosome. However, earlier experiments have documented a significantly (about 50%) lower absolute value |{Delta}Lk| than expected from the nucleosome geometry. Recently, using computer modeling, we have predicted two families of energetically stable conformations of the arrays with precisely positioned nucleosomes, one with an integer number of DNA turns in the linker DNA {L = 10n} and the other with extra five base pairs in the linker {L = 10n + 5}, to be topologically different. Here, using arrays of precisely positioned clone 601 nucleosomes, topological electrophoretic assays, and electron microscopy we experimentally tested these predictions. First, for small 12-mer nucleosome circular arrays we observed that dLk per nucleosome changes from -1.4 to -0.9 for the linkers {L = 10n} and {L = 10n + 5}, respectively. Second, for larger hybrid arrays containing a mixture of positioned and non-positioned nucleosomes we found that changing the DNA linker length within the positioned arrays was sufficient to significantly alter the overall DNA topology fully consistent with our prediction. The observed topological polymorphism of the circular nucleosome arrays provides a simple explanation for the DNA topology in native chromatin with variable DNA linker length. Furthermore, our results may reflect a more general tendency of chromosomal domains containing active or repressed genes to acquire different nucleosome spacing to retain topologically distinct higher-order structures.

molecular biology↗