Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: NKIRTIL063 neoantigen screen
| PRJNA1068303 | ENA
Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: validation in CD8 T cells
| PRJNA1068078 | ENA
Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: validation in CD4 T cells and OVC190 TCR screen
Project description:The screening of a previously reported fluorescein labelled 10,000 member PNA encoded peptide library allowed information on the interaction between the peptide-ligands and the cell surface receptors to be extracted, identified new peptide ligands for cell surface receptors, and gave crucial information about consensus sequences. A novel indirect amplification of the PNA signal by amplification of the PNA-complementary DNA library was developed to screen PNA-encoded peptide library against D54, HEK293T, and HEK293T-CCR6 cells. This work generates a new approach to biological discovery and an expansion of modern microarray techniques. In addition, the microarray approach facilitates screening for differences in surface-receptor ligands and/or receptor expression between various cell types including diseased and normal cells.
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:Here we describe our work on the development of an ABPP screening platform for cysteine reactive covalent fragments against deubiquitinating enzymes (DUBs) on 96 well plate format. Within this targeted workflow, we profile cysteine reactive fragments by competition with biotinylated ubiquitin probe in cell lysates and employ label free data independent acquisition (DIA) MS method. The platform was used to screen a library of 138 Cys-reactive covalent fragments against DUBome in HEK293T cell lysate, identifying functionally relevant hit fragments for numerous DUBs and demonstrating the utility of the approach in expanding the liganded proteome.
Project description:Here we describe our work on the development of an ABPP screening platform for cysteine reactive covalent fragments against deubiquitinating enzymes (DUBs) on 96 well plate format. Within this targeted workflow, we profile cysteine reactive fragments by competition with biotinylated ubiquitin probe in cell lysates and employ label free data independent acquisition (DIA) MS method. The platform was used to screen a library of 138 Cys-reactive covalent fragments against DUBome in HEK293T cell lysate, identifying functionally relevant hit fragments for numerous DUBs and demonstrating the utility of the approach in expanding the liganded proteome.
Project description:Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes raises the need for mechanisms to limit inappropriate gene regulation. DNA elements known as insulators provide one such mechanism by shielding promoters from an enhancer when placed in between these two elements. Whereas promoters and enhancers have been extensively characterized in animal genomes, insulators have not because of the lack of a high-throughput screening assay. In the absence of unbiased insulator screening in a genome, basic questions remain about the identity, density and diversity of genomic insulators. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila tissue culture cells to perform the first unbiased screen for insulators in any genome. By screening developmental gene loci, we found that not all characterized insulator protein binding sites are able to block enhancer-promoter communication. By dissecting the sequence determinants of functional insulators, we found that an unexpectedly broad sequence context around the central insulator protein binding motif is required for functionality. The ability to screen millions of DNA sequences in parallel without any positional effect has enabled the first functional mapping of insulators and provided further insights into the determinants of functional insulators.