Biology subjects

Zou, R. S.

Publications and source records attributed to Zou, R. S..

4 recordsLinked to original sources

bioRxiv2025

Cohesin drives chromatin scanning during the RAD51-mediated homology search.

Cohesin folds genomes into chromatin loops, whose roles are under debate. We report that double strand breaks (DSB) induce de novo formation of chromatin loops, with the break positioned at the loop base. These loops form only in S/G2 phases and occur during repair via homologous recombination (HR), concomitant with DNA end resection and RAD51 assembly. RAD51 showed two-tiered accumulation around DSBs, with a broad (~Mb) domain arising from the homology search. This domain is regulated by cohesin unloader, is constrained by TAD boundaries, and it overlaps with chromatin regions reeled through the break-anchored loop, suggesting that loop extrusion regulates the homology search. Indeed, depletion of NIPBL results in reduced HR, and this effect is more pronounced when the HR donor is far (~100 kb) from the break. Our data indicates that loop-extruding cohesin promotes the mammalian homology search by facilitating break-chromatin interactions within the damaged TAD. One-Sentence SummaryHigh spatiotemporal resolution analysis of double strand beak repair in 3D genome revealed the role of cohesin-driven loop extrusion in the homology search.

cell biology↗

bioRxiv2022

Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

Discovery of off-target CRISPR-Cas genome editing activity in patient-derived cells and animal models is crucial for therapeutic applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) accumulates repair protein MRE11 at CRISPR-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation sequencing (ChIP-seq). This technique, termed DISCOVER-Seq+, discovered up to 5-fold more CRISPR off-target sites in immortalized cell lines, primary human cells, and mice compared to previous methods. We demonstrated applicability to ex vivo knock-in of a cancer-directed transgenic T-cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. DISCOVER-Seq+ is the most sensitive method to-date for discovering off-target genome editing in vivo.

bioengineering↗

bioRxiv2022

Achieving single nucleotide sensitivity in direct hybridization genome imaging

Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, sgGOLDFISH, which leverages the high cleavage specificity of enhanced Cas9 combined with a single extended guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. Using sgGOLDFISH, we identified base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validated the identification through progerin immunofluorescence, and demonstrated accurate sub-nuclear localization of point mutations.

genomics↗

bioRxiv2022

Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites

We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

cell biology↗