Biology subjects

Urnov, F.

Publications and source records attributed to Urnov, F..

3 recordsLinked to original sources

bioRxiv2026

Reprogramming Cas9 PAM Recognition for Allele-Specific Editing

The therapeutic potential of CRISPR-Cas9 genome editing is fundamentally constrained by the requirement for specific short DNA sequences (PAMs) flanking the target site, limiting access to many clinically relevant genomic loci. This stringent PAM requirement is particularly problematic in applications which require precise positioning, such as base editing and allele-specific editing. Although PAM-relaxed variants have expanded the targetable genome, they incur trade-offs in on-target activity, off-target editing, and cleavage kinetics. This highlights an unmet need for variants that are re-targeted to alternative PAMs in order to maintain the specificity and enzymatic performance inherent to stringent dinucleotide PAM recognition. To overcome these limitations, we developed a yeast selection platform to engineering SpCas9 variants with re-specified PAM recognition. Using a clinically relevant Huntingtons disease gene (HTT) SNP as a proof-of-concept target, we engineered variants with reciprocal NGC and NGT PAM selectivity, as a step toward allele-specific editing in a large percentage of Huntingtons disease patients. These yeast-selected SpCas9 variants retained their modified activity across multiple endogenous HEK293T loci, demonstrating that this specificity is robust across diverse genomic contexts. The variants surpassed PAM-broadened variants on their respective on-target PAM while displaying broad loss of activity across alternative PAMs, effectively re-specifying PAM recognition toward a single dinucleotide sequence. Retargeted variants recovered on-target cleavage kinetics approaching that of wild-type SpCas9, even under competing substrate conditions, demonstrating that PAM re-specification can simultaneously restore catalytic efficiency and improve specificity. Beyond NGC and NGT, we leveraged our high-throughput platform to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.

molecular biology↗

bioRxiv2026

Phenotypic CRISPR screening identifies ZBTB10 as a novel regulator of human trophoblast differentiation

The human placenta is built by trophoblast cells that fuse together, secrete hormones, and invade the uterus, and defects in these processes contribute to pregnancy disorders such as preeclampsia. Because cell-cell fusion and hormone secretion are inherently non-cell-autonomous processes, their regulators have remained inaccessible to conventional pooled CRISPR screens. Here, we developed an arrayed CRISPR screen in fusogenic BeWo trophoblasts that simultaneously quantifies fusion and hCG secretion across 412 gene perturbations. The screen revealed that these two hallmark functions of trophoblast differentiation are genetically separable. We characterized the strongest novel hit, ZBTB10, in trophoblast stem cells, organoids, and placental tissue and find that ZBTB10 is an essential regulator of human trophoblast differentiation. ZBTB10 is required for invasive extravillous trophoblast differentiation and supports syncytiotrophoblast maturation, establishing it as a cross-lineage regulator that both activates and represses distinct trophoblast fate programs. Together, these findings provide a genetic platform and phenotypic dissection of how regulatory networks control human placental development.

cell biology↗

bioRxiv2025

Integrated analysis of multimodal long-read epigenetic assays

Long-read sequencing assays that detect base modifications are becoming increasingly important research tools for the study of epigenetic regulation, especially with the development of DiMeLo-seq and similar methods that deposit non-native base modifications to mark a range of epigenetic features such as protein-DNA interactions and chromatin accessibility. A main benefit of these methods is their inherent capacity for multimodality, enabling the encoding of multiple genomic signals onto single nucleic acid molecules. However, there are limited tools available for visualization and statistical analysis of this type of multimodal data. Here we introduce dimelo-toolkit, a python package built to enable flexible visualizations and easy integration into custom data processing workflows. We demonstrate the utility of dimelo-toolkits preset visualizations of multiple base modifications in long-read single-molecule sequencing data with a novel extension of the DiMeLo-seq protocol that can capture three separate aspects of chromatin state on the same single reads: target protein binding, CpG methylation, and chromatin accessibility. We apply this multimodal method to simultaneously map chromatin accessibility, CpG methylation, and LMNB1 and CTCF binding patterns, respectively, in GM12878 cells. Our flexible design allows us to investigate previously unexplored technical biases that arise when working with this type of multimodal data. Additionally, we show that dimelo-toolkit enables analysis for a wide range of other long-read sequencing methods, such as mapping endogenous patterns in RNA base modifications with direct RNA sequencing. This software tool will pave the way for developing well-optimized protocols and help unlock previously inaccessible biological insights.

genomics↗