Single-Macromolecule Studies of Eukaryotic Genomic Maintenance.

Genomes are self-organized and self-maintained as long, complex macromolecules of chromatin. The inherent heterogeneity, stochasticity, phase separation, and chromatin dynamics of genome operation make it challenging to study genomes using ensemble methods. Various single-molecule force-, fluorescent-, and sequencing-based techniques rooted in different disciplines have been developed to fill critical gaps in the capabilities of bulk measurements, each providing unique, otherwise inaccessible, insights into the structure and maintenance of the genome. Capable of capturing molecular-level details about the organization, conformational changes, and packaging of genetic material, as well as processive and stochastic movements of maintenance factors, a single-molecule toolbox provides an excellent opportunity for collaborative research to understand how genetic material functions in health and malfunctions in disease. In this review, we discuss novel insights brought to genomic sciences by single-molecule techniques and their potential to continue to revolutionize the field-one molecule at a time.

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Engineering a protein variant with a desired role relies on deep knowledge of the relationship between a protein's native structure and function. Using our structural understanding of a regulatory subdomain found in a family of DNA helicases, we engineered novel helicases for which the subdomain orientation is designed to switch between unwinding-inactive and -active conformations upon trans-cis isomerization of an azobenzene-based crosslinker. This on-demand light-based conformational control directly alters helicase activity as demonstrated by both bulk phase experiments and single-molecule optical tweezers analysis of one of the engineered helicases. The "opto-helicase" may be useful in future applications that require spatiotemporal control of DNA hybridization states.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma.

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.