Project description:The continuous evolution of SARS-CoV-2 poses global health challenges. A safe, rapid, and versatile method for assessing functions of Spike protein mutations in ACE2 receptor binding and immune evasion would be highly valuable. To address this, we engineered a transcription- and replication-competent virus-like particle (trVLP) derived from the Sindbis virus, pseudotyped with the SARS-CoV-2 receptor binding domain (RBD). This trVLP exclusively propagates in BHK-21 cell engineered to express both RNA replicase and human ACE2, providing a controllable, safe model of SARS-CoV-2 RBD-ACE2 interaction mediated virus entry. The system enables characterization of RBD interactions with ACE2 from various mammalian hosts, demonstrating its utility for studying host-virus interactions. By leveraging the evolutionary capability of trVLP mediated by error-prone RNA replication, we screened for RBD variants that evade the antibody-mediated inhibition of cell entry. Together, these findings underscore the utility of the trVLP as a safe, rapid, and flexible platform for dissecting SARS-CoV-2 RBD evolution and identifying key adaptive mutations with implications for surveillance and countermeasure development.
Project description:We developed a Ligation-based Library vs Library high-throughput Yeast Two-Hybrid (LLL-Y2H) screening system to explore protein interactions.
Project description:Fourth-generation EGFR tyrosine kinase are in development to overcome common resistance mutations. We performed deep mutational scanning (DMS) of the EGFR kinase domain in the context of L858R by introducing a saturation library of ~17,000 variants into Ba/F3 cells. DMS library-expressing cells were exposed to osimertinib or BLU-945 to identify escape mutations. L718X mutations were enriched across all conditions. BLU-945 specific mutations included K714R, K716T, L718V, T725M, K728E, K754E/N, N771S/T, T783I, Q791L/K, G863S, S895N, K929I, and M971L. A secondary DMS screen combining osimertinib and BLU-945 exclusively enriched for L718X mutations. Clinically, L718X mutations emerged in two patients treated with BLU-945. One patient with baseline EGFR L858R and L718Q mutations experienced early progression. Another with baseline EGFR L858R, T790M, and C797S acquired an L718V mutation at progression. This study demonstrate how comprehensive resistance profiling of targeted therapies can predict clinically relevant mutations and guide rational combinations to delay or prevent resistance.
Project description:Here we developed a massively parallel in-library ligation methodology to simultaneously perturb four pre-designed targets in CRISPR/Cas9 screening. Thousands of pairs of sequences precisely ligated with their counterparts in library, which enabled simultaneous expression of four gRNAs from each single vector. We demonstrated this novel method with 6,236 4-gene combinations targeting 1,599 immune response related genes, and generated a plasmid library with 1,400x coverage. The library performance was evaluated in a canonical T cell activation experiment, and combinations involved in TCR signaling pathway or TCR complex were successfully identified as positive regulators. Novel combination that is reflecting a potential pathway crosstalk was also verified. This new methodology expands the capacity of the perturbation in CRISPR screening and provided a powerful tool for researches in broad fields to study the combinatorial outcomes from coordinated gene behaviors.
Project description:CRISPR/Cas12a-based combinational screening has shown remarkable potential in identifying genetic interactions. Here, we described an innovative method for combinational genetic screen with rapid constructing of dual-crRNA library by gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which was identified previously by us with strict PAM recognition and low off- targeting, to guarantee the fidelity and efficiency. The custom, pooled SOE crRNA array (SOCA) library for double knockout screen could be conveniently constructed in lab for widespread use and CeCas12a mediated high fidelity screen display good performances even under negative selection screen. By designing an SOCA dual-crRNA library which covered the most of kinase and metabolism-associated gene targets of FDA- approved drugs that were implicated in hepatocellular carcinoma (HCC) tumorigenesis, novel cross talks between the two gene sets were negatively selected out to synergistically inhibit HCC cell growth in vitro and in vivo and also validated by virtual double knockdown screening based on TCGA databases. Thus through our rapid, efficient and high fidelity double knockout screening system, it is very promising to systemically dig genetic interactions between multiple gene sets or synergistic combinations of FDA-approved drugs for clinical translational medicine in the future.
Project description:Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library
Project description:Recovery of hairpins targeting a known prostate cancer pathway testing the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.