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Results for “bioengineering”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2022

Live Cell Lineage Tracing of Dormant Cancer Cells

Breast cancer is a leading cause of global cancer-related deaths, and metastasis is the overwhelming culprit of poor patient prognosis. The most nefarious aspect of metastasis is dormancy, a prolonged period between primary tumor resection and relapse. Current therapies are insufficient at killing dormant cells; thus, they can remain quiescent in the body for decades until eventually undergoing a phenotypic switch, resulting in metastases that are more adaptable and more drug resistant. Unfortunately, dormancy has few in vitro models, largely because lab-derived cell lines are highly proliferative. Existing models address tumor dormancy, not cellular dormancy, because tracking individual cells is technically challenging. To combat this problem, we adapted a live cell lineage approach to find and track individual dormant cells, distinguishing them from proliferative and dying cells over multiple days. We applied this approach across a range of different in vitro microenvironments. Our approach revealed that the proportion of cells that exhibited long-term quiescence was regulated by both cell intrinsic and extrinsic factors, with the most dormant cells found in 3D collagen gels. We hope this approach will prove useful to biologists and bioengineers in the dormancy community to identify, quantify, and study dormant tumor cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/511405v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@5d0af9org.highwire.dtl.DTLVardef@7b7830org.highwire.dtl.DTLVardef@f9fc4org.highwire.dtl.DTLVardef@1c20a39_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2022

Endonuclease fingerprint indicates a synthetic origin of SARS-CoV-2

To prevent future pandemics, it is important that we understand whether SARS-CoV-2 spilled over directly from animals to people, or indirectly in a laboratory accident. The genome of SARS-COV-2 contains a peculiar pattern of unique restriction endonuclease recognition sites allowing efficient dis- and re-assembly of the viral genome characteristic of synthetic viruses. Here, we report the likelihood of observing such a pattern in coronaviruses with no history of bioengineering. We find that SARS-CoV-2 is an anomaly, more likely a product of synthetic genome assembly than natural evolution. The restriction map of SARS-CoV-2 is consistent with many previously reported synthetic coronavirus genomes, meets all the criteria required for an efficient reverse genetic system, differs from closest relatives by a significantly higher rate of synonymous mutations in these synthetic-looking recognitions sites, and has a synthetic fingerprint unlikely to have evolved from its close relatives. We report a high likelihood that SARS-CoV-2 may have originated as an infectious clone assembled in vitro. Lay SummaryTo construct synthetic variants of natural coronaviruses in the lab, researchers often use a method called in vitro genome assembly. This method utilizes special enzymes called restriction enzymes to generate DNA building blocks that then can be "stitched" together in the correct order of the viral genome. To make a virus in the lab, researchers usually engineer the viral genome to add and remove stitching sites, called restriction sites. The ways researchers modify these sites can serve as fingerprints of in vitro genome assembly. We found that SARS-CoV has the restriction site fingerprint that is typical for synthetic viruses. The synthetic fingerprint of SARS-CoV-2 is anomalous in wild coronaviruses, and common in lab-assembled viruses. The type of mutations (synonymous or silent mutations) that differentiate the restriction sites in SARS-CoV-2 are characteristic of engineering, and the concentration of these silent mutations in the restriction sites is extremely unlikely to have arisen by random evolution. Both the restriction site fingerprint and the pattern of mutations generating them are extremely unlikely in wild coronaviruses and nearly universal in synthetic viruses. Our findings strongly suggest a synthetic origin of SARS-CoV2.

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bioRxiv2022

DNA origami directed virus capsid polymorphism

Most known viruses protect their genome by encapsulating it inside a protein capsid. Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. The assembly process of native capsids is highly cooperative and governed by the protein geometry, protein-protein as well as protein-nucleic acid interactions. Importantly, the absolute control over the size and shape of virus capsids would have imminent advantages in the development of new vaccines and delivery systems. However, tools to direct the assembly process in a programmable manner are exceedingly elusive or strictly limited to specific structures. Here, we introduce a modular approach by demonstrating DNA origami directed polymorphism of single protein subunit capsids. We achieve control over the capsid shape, size, and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms for the capsid proteins. Binding assays and single-particle cryo-electron microscopy reconstruction show that the DNA origami nanoshapes are efficiently encapsulated within the capsid. Further, we observe that helical arrangement of hexameric capsomers is the preferred mode of packing, while a negative curvature of the origami structure is not well tolerated. The capsid proteins assemble on DNA origami in single or double layer configurations depending on the applied stoichiometry. In addition, the obtained viral capsid coatings are able to efficiently shield the encapsulated DNA origami from nuclease degradation and prevent the structures from aggregation. Therefore, these findings may in addition find direct implementations in DNA nanotechnology-based bioengineering by paving the way for the next-generation cargo protection and targeting strategies.

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bioRxiv2022

Inflammatory Synovial Fibroblast Culture in 3D Systems: A Comparative Transcriptomic and Functional Study.

Inflammation is essential for fighting infections and initiating tissue repair, but chronic unresolved inflammation underlies many conditions, like cancer and autoimmune disorders. While dysregulated immune responses drive chronic inflammation, non-immune stromal cells such as fibroblasts also play a critical role. Targeting fibroblasts could enable tissue-specific therapies while avoiding the systemic suppression caused by current drugs. However, traditional culture systems often induce artificial behaviours, limiting progress. Here, we demonstrate the importance of the mechanical properties of the 3D culture environments in fibroblast-mediated inflammation in the context of Rheumatoid Arthritis, a chronic disease that primarily affects joints but also impacts other organs. We isolated fibroblasts from healthy and arthritic mouse joints and expanded them on 2D tissue culture plastic, stiff fibronectin-coated scaffolds, or soft pegylated fibronectin-based hydrogels. Our results highlight the plasticity of fibroblasts, with microenvironmental cues driving inflammatory or regulatory phenotypes. The 3D environment offered by fibronectin-coated scaffolds restored inflammatory gene expression profiles that were lost in flat cultures, while the more physiologically relevant soft hydrogels shifted fibroblasts toward a resolving phenotype. These findings underscore the importance of the 3D environment in modulating fibroblast behaviour and establish a foundation for bioengineered systems that better model disease or guide therapeutic strategies.

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bioRxiv2022

Cholesterol-deficient T cell membrane-encapsulated STING agonists for tumor-targeted immunotherapy

In recent years, STING agonists have shown promising results in enhancing tumor immunotherapies. Nanoparticle-based tumor targeting delivery of STING agonists is considered as an important way to improve the therapeutic efficacy and reduce the side effects of STING agonists. However, how to escape the clearance to nanoparticles by phagocytes in the blood while maintaining the tumor targeting efficiency of nanoparticles is still a big challenge. Herein, cholesterol-deficient membrane from bioengineered T cells overexpressing PD-1 encapsulated STING agonist SR-717 (a.k.a. COMs) was used to treat melanoma. Nanoparticles coated by these membranes displayed remarkably dropped clearance by monocytes in the blood in both animal and human blood comparing with nanoparticles coated by non-modified T cell membrane, while maintaining the high tumor cell targeting efficiency of COMs. In mice melanoma model, intravenous injected COMs successfully delivered SR-717 to tumor and activated STING pathways and the PD-1 on COMs blocked the up-regulated PD-L1 in tumor cells induced by SR-717. As a result, COMs stimulated strong tumor immune responses to inhibit melanoma recurrence when it combined with photothermal therapy (PTT). In summary, this study developed a highly effective bionic system that integrated STING activation and immunotherapy, and provided a simple and effective strategy to enhance performance of cell membrane-coated delivery systems in vivo.

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bioRxiv2023

Quiescence-inducing 3D-engineered matrix uncovers mechanosensitive and drug protective FHL2-p21 signaling axis

Resected tumors frequently relapse with distant metastasis, despite systemic treatment. Cellular dormancy has been identified as an important mechanism underlying such drug resistance enabling late relapse. Nonetheless, hurdles associated with detection and isolation of disseminated cancer cells (DCCs) in disease-free patients urge the need for in vitro models of dormant cells suited for drug discovery. Here, we explore dormancy-inducing 3D-engineered matrices, which generate mechanical confinement and induce growth arrest and survival against chemotherapy in cancer cells. We characterized the dormant phenotype of solitary cells by P-ERKlow:P-p38high dormancy signaling ratio, along with Ki67-expression. As underlying mechanism, we identified stiffness-dependent nuclear localization of the four-and-a-half LIM domains 2 (FHL2) protein, leading to p53-independent high p21Cip1/Waf1 nuclear expression, validated in murine and human tissue. Suggestive of a resistance-causing role, cells in the dormancy-inducing matrix became sensitive against chemotherapy upon FHL2 downregulation. Thus, our biomaterial-based approach will enable systematic screens for novel compounds suited to eradicate potentially relapsing, dormant cancer cells. TeaserUsing semi-synthetic bioengineered hydrogels, we reveal a mechanosensitive and drug protective mechanism of dormant cancer cells in tissues

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bioRxiv2023

Phenotypically Complex Living Materials Containing Engineered Cyanobacteria

A cyanobacterial photosynthetic biocomposite material was fabricated using 3D-printing and bioengineered to produce multiple functional outputs in response to an external chemical stimulus. Our investigations show the advantages of utilizing additive manufacturing techniques in controlling the design and shape of the fabricated materials, which proved to be important for the support and growth of obligate phototrophic microorganisms within the material. As an initial proof-of-concept, a synthetic theophylline-responsive riboswitch in Synechococcus elongatus PCC 7942 was used for regulating the expression of a yellow fluorescent protein (YFP) reporter. Upon induction with theophylline, the encapsulated cells produced YFP within the hydrogel matrix. Subsequently, a strain of S. elongatus was engineered to produce an oxidative enzyme that is useful for bioremediation, laccase, expressed either constitutively or under the control of the riboswitch. The responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a useful tool in environmental bioremediation. Finally, cells were engineered to have the capacity for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing unintended environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in patterned volumetric 3D-printed designs, we demonstrate the potential of programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

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bioRxiv2023

Programmable RNA detection with CRISPR-Cas12a

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or SAHARA to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes. O_FIG O_LINKSMALLFIG WIDTH=125 HEIGHT=200 SRC="FIGDIR/small/525716v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@5edb2dorg.highwire.dtl.DTLVardef@187678borg.highwire.dtl.DTLVardef@9f0184org.highwire.dtl.DTLVardef@11cb1a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2023

Synthetic cell armor made of DNA origami

Therapeutic and bioengineering applications of cells, such as cell printing and cell delivery, are directly limited by cell damage and death due to harsh mechanical conditions. Improved cellular robustness thus motivates investigations into cell encapsulation that provides essential protection. Here we target the cell-surface glycocalyx and crosslink two layers of DNA origami nanorods on the cellular plasma membrane to form a nanoscale protective shell. This modular and programmable approach enables fine control over the layering and composition of membrane-deposited nanorods. We show that the DNA origami nanoshell modulates the biophysical properties of cell membranes by enhancing membrane stiffness and lowering lipid fluidity. Moreover, the nanoshell serves as armor, protecting cells, limiting swelling and ultimately improving their viability against mechanical stress from osmotic imbalance and centrifugal forces. Our results demonstrate the potential of the nanoshell, not only as a cellular protection strategy, but also as a platform for manipulating and studying plasma membrane mechanics.

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bioRxiv2023

Self-organization of Long-lasting Human Endothelial Capillary Networks guided by DLP Bioprinting

Tissue engineering holds great promise for regenerative medicine, drug discovery and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential and is a major challenge to be met. In this work, we explore a promising Digital Light Processing (DLP) bioprinting approach to encapsulate endothelial progenitor cells (EPCs) in 3D photopolymerized hydrogel scaffolds to guide them towards vascular network formation. We observed that EPCs encapsulated in the appropriate photopolymerized hydrogel can proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of various diameters (in the range of 10 to 100 m). Presenting a monolayer wall of endothelial cells strongly connected by tight junctions around a central lumen, these structures can be microinjected with fluorescent dye and are stable for several weeks in vitro. Interestingly, our technology has proven to be versatile in promoting the formation of vascular structures using a variety of vascular cell lines, including EPCs, human vascular endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs). We have also demonstrated that these vascular structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This opens new opportunities for future applications, such as stacking these endothelial vascular structures with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

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bioRxiv2023

Novel endogenous engineering platform for robust loading and delivery of functional mRNA by extracellular vesicles

Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a plethora of clinical applications. For in vivo functionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, a bioengineering platform for efficient mRNA loading and functional delivery using bionormal nanoparticles, Extracellular Vesicles (EVs), is established by expressing a highly specific RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA. The additional combination with a fusogenic endosomal escape moiety, VSVg, enables functional mRNA delivery in vivo at doses substantially lower than currently used clinically with synthetic lipid-based nanoparticles. Importantly, the application of EVs loaded with effective cancer immunotherapy proves highly effective in an aggressive melanoma mouse model. This technology addresses substantial drawbacks currently associated with EV-based nucleic acid delivery systems and is a leap forward to clinical EV applications. Graphical Abstract(Figure created using BioRender) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/533081v3_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@140058eorg.highwire.dtl.DTLVardef@2ceaccorg.highwire.dtl.DTLVardef@eafd52org.highwire.dtl.DTLVardef@19d4019_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2023

Colorectal cancer detection and treatment with engineered probiotics

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by [~]50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

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bioRxiv2023

Heart-on-a-chip model of immune-induced cardiac dysfunction reveals the involvement of free mitochondrial DNA and therapeutic effects of endothelial exosomes

Cardiovascular disease continues to take more human lives than all cancer combined, prompting the need for improved research models and treatment options. Despite a significant progress in development of mature heart-on-a-chip models of fibrosis and cardiomyopathies starting from induced pluripotent stem cells (iPSCs), human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip system with circulating immune cells to model SARS-CoV-2-induced acute myocarditis. Briefly, we observed hallmarks of COVID-19-induced myocardial inflammation in the heart-on-a-chip model, as the presence of immune cells augmented the expression levels of proinflammatory cytokines, triggered progressive impairment of contractile function and altered intracellular calcium transient activities. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the in vitro heart-on-a-chip model and then validated in COVID-19 patients with low left ventricular ejection fraction (LVEF), demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2 induced myocardial inflammation, we established that administration of human umbilical vein-derived EVs effectively rescued the contractile deficit, normalized intracellular calcium handling, elevated the contraction force and reduced the ccf- mtDNA and chemokine release via TLR-NF-kB signaling axis.

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bioRxiv2023

Multi-modal profiling of biostabilized human skin modules reveals a coordinated ecosystem response to injected mRNA-1273 COVID-19 vaccine.

The field of vaccination is witnessing a remarkable surge in the development of innovative strategies. There is a need to develop technological platforms capable of generating human data prior to progressing to clinical trials. Here we introduce VaxSkin, a flexible solution designed for the comprehensive monitoring of the natural human skin ecosystems response to vaccines over time. Based on bioengineering to repurpose surgical resections, it allows a comprehensive analysis of the response to vaccines at both organ and single-cell levels. Upon injection of the mRNA-1273 COVID-19 vaccine, we characterized precise sequential molecular events triggered upon detection of the exogenous substance. We also found that the vaccine consistently targets DC/macrophages and mast cells, regardless of the administration route, while promoting specific cell-cell communications in surrounding immune cell subsets. Given its direct translational relevance, VaxSkin provides a multiscale vision of skin vaccination that could pave the way toward the development of new vaccination development strategies.

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bioRxiv2023

Granular hydrogels improve myogenic invasion and repair after volumetric muscle loss

Skeletal muscle injuries including volumetric muscle loss (VML) are marked by excessive scarring and functional disability that inherent regenerative mechanisms are unable to reverse. Despite high prevalence in civilian and military populations, there is currently no effective treatment for VML but bioengineering interventions such as biomaterials that fill the VML defect to support tissue growth and repair are a promising strategy. However, traditional biomaterials developed for this purpose are rigid, non-porous constructs that hinder cell infiltration. In the present study, we test the effects of granular hydrogels on muscle repair - hypothesizing that their inherent porosity will support the invasion of native myogenic cells and their flowability will permit conformable filling of the defect site, leading to effective muscle repair. We used photocurable hyaluronic acid crosslinked with matched muscle stiffness to prepare small or large particle fragments via extrusion fragmentation and facile size sorting. In assembled granular hydrogels, particle size and degree of packing significantly influenced pore features including porosity, pore size, and pore density, as well as rheological behavior including storage moduli and yield strain. We tested the ability of granular hydrogels to support early-stage (satellite cell invasion) and late-stage (myofiber invasion) muscle repair compared to bulk hydrogels in a VML injury model in the tibialis anterior (TA) muscles of 12-14 week old mice. Histological evaluation revealed granular hydrogels supported these regenerative processes while control bulk hydrogels restricted them to the gel-tissue interface in line with the absence of invading cells. Together, these results highlight the promising potential of injectable and porous granular hydrogels in supporting endogenous repair after severe muscle injury.

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bioRxiv2023

Biodesign of Frugal CRISPR Kits for Equitable and Accessible Education

Equitable and accessible education on life sciences and bioengineering is crucial to training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology democratization that can benefit a wide-ranging population. As a ground-breaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology for educational purposes has remained restricted, primarily due to extensive resources required to execute CRISPR experiments. In this study, we develop an accessible and frugal CRISPR kit, tailored for K-12 education settings. Our CRISPR kit eliminates the need for equipment, prioritizes biosafety, and utilizes cost-effective reagents. By combining CRISPRi gene regulation, chromoproteins, cell-free transcription-translation systems, and smartphone-based quantification, our kit offers a user-friendly approach and a reliable assessment of CRISPR activity, eliminating the need for a traditional laboratory setup. Experiments conducted by high school students in real-world settings highlight the kits utility for conducting reliable CRISPR experiments. The frugal CRISPR kit provides a modular and expandable platform to offer hands-on experience in genome engineering, and will facilitate equitable and accessible education and technology democratization for communities of diverse socioeconomic and geographic backgrounds.

bioengineering↗

bioRxiv2023

Extracellular matrix sulfation in the tumor microenvironment stimulates cancer stemness and invasiveness

Tumor extracellular matrices (ECM) exhibit aberrant changes in composition and mechanics compared to normal tissues. Proteoglycans (PG) are vital regulators of cellular signaling in the ECM with ability to modulate receptor tyrosine kinase (RTK) activation via their sulfated glycosaminoglycan (sGAG) side chains. However, their role on tumor cell behavior is controversial. Here, we demonstrate that PGs are heavily expressed in lung adenocarcinoma patients in correlation with invasive phenotype and poor prognosis. We developed a bioengineered human lung tumor model which recapitulates the increase of sGAGs in tumors in an organotypic matrix with independent control of stiffness, viscoelasticity, ligand density and porosity. Our model reveals that increased sulfation stimulates extensive proliferation, epithelial-mesenchymal transition and stemness in cancer cells. We identified the FAK-PI3K-mTOR signaling axis as a mediator of sulfation-induced molecular changes in cells upon activation of a distinct set of RTKs within tumor-mimetic hydrogels. We demonstrate that the transcriptomic landscape of tumor cells in response to increased sulfation resembles native PG-rich patient tumors through employing integrative omics and network modeling approaches.

bioengineering↗

bioRxiv2023

Protein Engineering with Lightweight Graph Denoising Neural Networks

Protein engineering faces challenges in finding optimal mutants from the massive pool of candidate mutants. In this study, we introduce a deep learning-based data-efficient fitness prediction tool to steer protein engineering. Our methodology establishes a lightweight graph neural network scheme for protein structures, which efficiently analyzes the microenvironment of amino acids in wild-type proteins and reconstructs the distribution of the amino acid sequences that are more likely to pass natural selection. This distribution serves as a general guidance for scoring proteins toward arbitrary properties on any order of mutations. Our proposed solution undergoes extensive wet-lab experimental validation spanning diverse physicochemical properties of various proteins, including fluorescence intensity, antigen-antibody affinity, thermostability, and DNA cleavage activity. More than 40% of PO_SCPLOWROTC_SCPLOWLGN-designed single-site mutants outperform their wild-type counterparts across all studied proteins and targeted properties. More importantly, our model can bypass the negative epistatic effect to combine single mutation sites and form deep mutants with up to 7 mutation sites in a single round, whose physicochemical properties are significantly improved. This observation provides compelling evidence of the structure-based models potential to guide deep mutations in protein engineering. Overall, our approach emerges as a versatile tool for protein engineering, benefiting both the computational and bioengineering communities.

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