Biology subjects

Shah, S. T.

Publications and source records attributed to Shah, S. T..

4 recordsLinked to original sources

bioRxiv2026

Structure and antigenicity of the 2026 Bundibugyo virus glycoprotein

An outbreak of ebolavirus disease caused by a new zoonotic spillover of Bundibugyo virus (BDBV) is currently spreading at an unprecedented pace in the Democratic Republic of the Congo and Uganda. The outbreak has resulted in 7,733 cases with a case-fatality ratio of 48%, yet no BDBV-specific vaccines or therapeutics are available. The ebolavirus glycoprotein (GP) promotes viral entry and is the target of neutralizing antibodies and vaccine development. Here, we engineered a soluble, prefusion-stabilized GP trimer from a contemporary BDBV isolate and determined its cryo-electron microscopy structure bound to the broadly neutralizing antibody EBOV-515. It reveals the molecular basis for EBOV-515 cross-reactivity and suggests that the antibody inhibits viral entry by interfering with NPC1 engagement in addition to trapping GP in the prefusion conformation and hindering glycan cap cleavage. Mapping lineage-defining substitutions identified changes in GP1 and GP2, including R506S within an antigenic site targeted by multiple broadly neutralizing antibodies recognizing the fusion loop. Nevertheless, we found that several glycan cap-, GP base-, and HR2-directed antibodies cross-reacted and neutralized BDBV DRC/2026 GP pseudovirus with potency comparable or greater to that observed against earlier BDBV isolates and the Zaire ebolavirus Makona/2024 isolate. These findings define the antigenic properties of contemporary BDBV GP and delineate conserved epitopes as ideal targets for antibody therapeutics and for the development of pan-ebolavirus vaccines.

biochemistry↗

bioRxiv2026

Optimized parameters for Cas9 CRISPR interference library design

CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific sgRNAs for the design of compact libraries. While several genome-wide Cas9 CRISPRi libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed sequence patterns. We leverage these findings to design an optimized Cas9 CRISPRi library, Katsano, and validate its performance with genome-wide viability screens.

bioengineering↗

bioRxiv2025

Balancing off-target and on-target considerations for optimized Cas9 CRISPR knockout library design

The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations yield library deprecation over time, further motivating an updated genome-wide design effort. Recently, we have developed an enhanced model, Rule Set 3, which leveraged an expansive training set and feature space to predict guide efficacy. However, the benefit of such advances to library design is limited by current approaches to balance predictions of on-target activity with off-target considerations. Here we present a guide selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides. We pair this model with strategic design choices to create Jacquere, an updated, optimized, and validated Cas9 CRISPR knockout (CRISPRko) genome-wide library for the human genome.

genomics↗

bioRxiv2024

Activity-based selection for enhanced base editor mutational scanning

Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome, which is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here, we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.

genetics↗