Engineered FnCas12a with enhanced activity through directional evolution in human cells.
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ABSTRACT:
SUBMITTER: Liu X
PROVIDER: S-EPMC7961096 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted toward the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high-throughput screen was implemented to functionally interrogate combinatorial libraries of charge-engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge-engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.
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Project description:Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.
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Project description:Human T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-? (FR?) have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of their inability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FR?-specific scFv (MOv19) coupled to the T-cell receptor CD3? chain signaling module alone (MOv19-?) or in combination with the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BB?). Primary human T cells transduced to express conventional MOv19-? or costimulated MOv19-BB? CARs secreted various proinflammatory cytokines, and exerted cytotoxic function when cocultured with FR?(+) tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BB? CAR mediated tumor regression in immunodeficient mice bearing large, established FR?(+) human cancer. MOv19-BB? CAR T-cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BB? CAR T cells. T-cell persistence and antitumor activity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cell persistence but not antitumor activity in vivo. Our results show that anti-FR? CAR outfitted with CD137 costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limit the conventional FR? T-cell targeting strategy to provide potent antitumor activity in vivo.
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Project description:Genetically engineered organisms are prone to evolve in response to the engineering. This evolution is often undesirable and can negatively affect the purpose of the engineering. Methods that maintain the stability of engineered genomes are therefore critical to the successful design and use of genetically engineered organisms. One potential method to limit unwanted evolution is by taking advantage of the ability of gene flow to counter local adaption, a process of supplementation. Here, we investigate the feasibility of supplementation as a mechanism to offset the evolutionary degradation of a transgene in three model systems: a bioreactor, a gene drive, and a transmissible vaccine. In each model, continual introduction from a stock is used to balance mutation and selection against the transgene. Each system has its unique features. The bioreactor system is especially tractable and has a simple answer: The level of supplementation required to maintain the transgene at a frequency p^ is approximately p^s , where s is the selective disadvantage of the transgene. Supplementation is also feasible in the transmissible vaccine case but is probably not practical to prevent the evolution of resistance against a gene drive. We note, however, that the continual replacement of even a small fraction of a large population can be challenging, limiting the usefulness of supplementation as a means of controlling unwanted evolution.
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Project description:Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.
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Project description:Directed cell migration by contact guidance in aligned collagenous extracellular matrix (ECM) is a critical enabler of breast cancer dissemination. The mechanisms of this process are poorly understood, particularly in 3D, in part because of the lack of efficient methods to generate aligned collagen matrices. To address this technological gap, we propose a simple method to align collagen gels using guided cellular compaction. Our method yields highly aligned, acellular collagen constructs with predictable microstructural features, thus providing a controlled microenvironment for in vitro experiments. Quantifying cell behavior in these anisotropic constructs, we find that breast carcinoma cells are acutely sensitive to the direction and extent of collagen alignment. Further, live cell imaging and analysis of 3D cell migration reveals that alignment of collagen does not alter the total motility of breast cancer cells, but simply redirects their migration to produce largely one-dimensional movement. However, a profoundly enhanced motility in aligned collagen matrices is observed for the subpopulation of carcinoma cells with high tumor initiating and metastatic capacity, termed cancer stem cells (CSCs). Analysis of the biophysical determinants of cell migration show that nuclear deformation is not a critical factor associated with the observed increases in motility for CSCs. Rather, smaller cell size, a high degree of phenotypic plasticity, and increased protrusive activity emerge as vital facilitators of rapid, contact-guided migration of CSCs in aligned 3D collagen matrices.
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Project description:We assess the prevalence of polygenic evolution in pathways between the yeasts S. cerevisiae and S. bayanus. We first established short-read sequencing methods to detect cis-regulatory variation in a diploid hybrid between the species. We then formulated an analytic strategy to test for the scenario in which selective pressure in one species, to increase or decrease the activity of a pathway, has driven the accumulation of cis-regulatory variants that act in the same direction on gene expression. Application of this test revealed a variety of yeast pathways with evidence for directional regulatory evolution. Measurement of allele-specific expression of each ortholog in a diploid hybrid between Saccharomyces species
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