Howard Chang, Doctoral Dissertation Advisor (AC)
CRISPR-engineered T cells in patients with refractory cancer.
Science (New York, N.Y.)
CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase I clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC) were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding PD-1 (PDCD1), was performed to improve anti-tumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Though chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene-editing for cancer immunotherapy.
View details for DOI 10.1126/science.aba7365
View details for PubMedID 32029687
Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
View details for DOI 10.1016/j.cell.2019.05.027
View details for PubMedID 31230715
Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.
Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in 4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human Blymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFsuncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
View details for PubMedID 30580963
Transcript-indexed ATAC-seq for precision immune profiling.
T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.
View details for PubMedID 29686426
ciRS-7 exonic sequence is embedded in a long non-coding RNA locus
2017; 13 (12): e1007114
ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. To study the biogenesis of ciRS-7, we developed an algorithm to define its promoter and predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generates a new model for regulation of this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChIP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.
View details for PubMedID 29236709