Cell Reports Physical Science

Gene therapy studies have been of great importance in the elimination of genetic diseases, and the capability of the CRISPR/Cas9 genome editing technique to correct genetic defects has shown great promise. As DNA-based Cas9 nuclease delivery is preferable because of its low cost and higher stability, effective vector-based CRISPR/Cas9 administration is urgently needed. Here, we used the multicellular organism Caenorhabditis elegans to optimize the polymer-mediated DNA delivery system to generate mutants with CRISPR/Cas9. Toward this end, the cationically quaternized polymer of POEGMA-b-P4VP (POEGMA-b-QP4VP) as a carrier of CRISPR/Cas9 components was first synthesized, followed by the formation of plasmid DNA-polymer complex called polyplexes. 1H NMR, Zeta-Sizer, Scanning Electron Microscopy (SEM) analysis, and gel retardation experiments confirmed the polyplexes formation, including pRF4 (Roller) and sgRNA dpy-10, which were then incubated with C. elegans. The polymer-mediated delivery system facilitated the generation of transgenic Roller animals and heritable Dumpy mutants with CRISPR/Cas9. Our study for the first time demonstrated optimized administration of CRISPR/Cas 9 components to C. elegans.

During repetitive bending of cilia and flagella, axonemal dynein molecules move in an oscillatory manner along a microtubule (MT), but how the minus-end-directed motor dynein can oscillate back and forth is unknown. There are various factors that may regulate the dynein activities, e.g., the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of the oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and DNA origami that crosslinks the MTs. Electron microscopy (EM) showed patches of dynein molecules crossbridging two MTs in two opposite orientations; the oppositely oriented dyneins are expected to produce opposing forces. The optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillate back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without the additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

CRISPR-Cas systems have revolutionized genome editing with their precision and versatility, enabling transformative applications in various fields, especially in the treatment of genetic diseases. However, the clinical translation of this technology is hindered by challenges such as off-target effects and uncontrolled nuclease activity. At the same time, it has the possibility of causing biosecurity risks, underscoring the urgent need for reliable regulatory tools. Existing CRISPR inhibitors, primarily anti-CRISPR protein or exogenously synthesized small molecules, are limited by their specificity or bioavailability and long research period, unable to address the diverse CRISPR nucleases used in research and therapy. Based on the phenomena obtained from various in vitro and cell experiments, combining molecular dynamics simulation and bio - layer interferometry (BLI) analysis, here we report a naturally occurring small-molecule {beta}-nicotinamide mononucleotide (NMN), the first known endogenous metabolite with broad-spectrum inhibitory activity against multiple CRISPR-associated proteins (Cas9, Cas12, and Cas13) through various mechanisms. Our findings establish NMN as a dual-purpose tool, which reduces cell damage caused by gene editing and mitigates risks of unintended genetic modifications in research and clinical settings. This discovery further shortens the distance between basic medicine and translational medicine, providing a new approach for developing endogenous regulatory molecules in genome engineering.

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, the genetic manipulation methods in tardigrades have long been desired. Despite our prior success in somatic cell gene-editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions conductive to generating gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRIPSR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. Through co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny and these edited-alleles were inherited by G1/G2 progeny. This establishment of a simple method for generating homozygous knock-out/knock-in individuals not only facilitates in vivo analyses of molecular mechanisms underpinning extreme tolerance but also opens avenues for exploring various topics, including Evo-Devo, in tardigrades.

Synthetic circular RNA (circRNA) has emerged as a promising platform for vaccine and therapeutic development, featuring its uniqueness in a closed-loop structure, cap-independent translation mechanism, and prolonged expression. However, the rational design of a circRNA sequence to jointly improve its stability and protein coding potential remains challenging. In this study, we present circDesign, an efficient algorithm to achieve the optimal design of circRNA by ensuring optimized folding of each segment, which leads to enhanced circularization efficiency, stability, and translatability. Using rabies virus glycoprotein (RABV-G) as the model antigen, we demonstrated that circDesign-generated circRNAs exhibited higher stability and protein translation efficiency in vitro and in vivo compared to other codon adaptation index (CAI)-optimized sequences, thus leading to enhanced in vivo immunogenicity. Ribosome and polysome profiling further revealed that an intact internal ribosome entry site (IRES) structure is critical for efficient translation. By intentionally disrupting the IRES motifs, we observed that the resulting circRNA sequences had lower translatability compared to circDesign-generated sequences. Taken together, our circular RNA design algorithm provides a general strategy to leverage the capability of circRNA as next-generation vaccines or therapeutics.

Nanoparticle-based drug delivery systems have the potential to revolutionize medicine but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at the in utero stage have the potential to overcome these key limitations, due to the high rate of angiogenesis and cell division in fetal tissue, and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA for gene editing enzymes in utero after an intrahepatic injection, and can access and edit major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero after an intrahepatic injection. These experiments demonstrate the possibility of non-viral delivery of gene editing enzymes in utero and nanoparticle drug delivery has great potential for delivering macromolecules to organs outside of the liver in utero, which provides a promising strategy for treating a wide variety of devastating genetic diseases before birth.

Double-stranded RNA (dsRNA) has evolved into a key tool in understanding and regulating biological processes, with promising implications in therapeutics. However, its efficacy is often limited due to instability in biological settings. Recently, the development of peptidic dsRNA binders derived from naturally occurring RNA-binding proteins has emerged as a favorable starting point to address this limitation. Nevertheless, it remains unclear how these high affinity dsRNA binders alter the structure and flexibility of dsRNA. To this end, we employed single-molecule magnetic tweezers experiments to investigate the effects of TAV2b-derived peptidic dsRNA binders on the mechanical properties of dsRNA. Torque spectroscopy assays demonstrated that these peptides underwind dsRNA, while also stabilizing the duplex. Additionally, force spectroscopy experiments demonstrate that a wild type TAV2b peptide derivative extends the contour length and lowers the bending rigidity of dsRNA, while a homodimeric version triggers the formation of higher order complexes at forces below 1 pN. Our study presents a quantitative approach to investigate how these peptides alter the structure of dsRNA, and whether peptide structural design alters the affinity to dsRNA and its stability. This approach could inform the design of more potent and effective dsRNA binders in the efforts to advance RNA therapeutics.

The frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the [Formula] pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

The CRISPR/Cas9 system is now the gold standard for the generation of genetically modified cell and animal models but knockin is a bottleneck. One reason could be that there is no consensus regarding the concentrations of its components to be used. Here, we defined optimal Cas9 protein, guide RNA and short donor DNA concentrations on a GFP to BFP conversion model of human induced pluripotent stem cells and point mutations on rat transgenic embryos. With a molecular rational approach of the CRISPR/Cas9 system and study of ribonucleoprotein complex formation by nanodifferential scanning fluorimetry, we defined that Cas9/guide RNA 1/1 molar ratio with 0.2M and 0.4M of Cas9, coupled with 2M of ssODN are sufficient for optimal and high knockin frequencies in rat embryos and human induced pluripotent stem cells, respectively. These optimal conditions use lower concentrations of CRISPR reagents to form the RNP complex than most conditions published while achieving 50% of knockin. This study allowed us to reduce costs and toxicity while improving editing and knockin efficacy on two particularly key models to mimic human diseases.

The integration of artificial intelligence (AI) and machine learning (ML) in peptide design has revolutionized the development of antimicrobial peptides (AMPs), which are essential components of innate immunity. In this study, we identified a novel synthetic peptide, PAN4 (GAYTFKIRRK), through genetic screening of AI-generated candidates in Drosophila melanogaster. PAN4 demonstrated robust antimicrobial activity, stress tolerance, and antitumor effects, significantly enhancing survival rates following bacterial infections and improving locomotor behaviors without adversely affecting lifespan. Furthermore, PAN4 expression in intestinal stem cells completely suppressed RasV12-induced tumor progression, indicating its potential role in cancer prevention. The peptide also mitigated gut barrier dysfunction associated with sleep deprivation and reduced inflammation in a dextran sulfate sodium (DSS)-induced colitis model. Mechanistically, PAN4s antimicrobial activity was linked to its interaction with specific peptidoglycan recognition proteins (PGRPs), particularly PGRP-SC1a, while the Tak1-mediated immune signaling pathway was found to be non-essential for its efficacy. PAN4 showed promising effects on honeybee health, enhancing survival rates under bacterial stress. Furthermore, PAN4 expression demonstrated significant anti-tumor activity in the Drosophila gut tumor model. Our findings suggest that PAN4 serves as a versatile agent with significant implications for enhancing immune responses and combating diseases in honeybee populations, paving the way for future applications in agriculture and medicine.

DNA-encoded peptide libraries are ideal functional peptide discovery platforms for their extremely large capacity. However, its still difficult to build high content peptide library in intact mammalian cells, which offer advantages associated with appropriate protein modification, proper protein folding, and natural status of membrane protein. Our previous work established linear-double-stranded DNAs (ldsDNAs) as innovative biological parts to implement AND gate genetic circuits in mammalian cell line. In the current study, we employ ldsDNA with terminal NNK degenerate codons as AND gate input to build highly diverse peptide library in mammalian cells. This ldsDNA-based AND gate (LBAG) peptide strategy is easy to conduct, only PCR reaction and cell transfection experiments are needed. High-throughput sequencing (HTS) results reveal that our new LBAG strategy could generate peptide library with both amino acid sequence and peptide length diversities. Moreover, by a mammalian cell two-hybrid system, we pan an MDM2 protein interacting peptide through the LBAG peptide library. Our work establishes ldsDNA as biological parts for building highly diverse peptide library in mammalian cells.

Chitin is found in the exoskeleton and peritrophic matrix of arthropods, but recent studies have also identified chitin in the spinning duct of silk-spinning arthropods. Here, we report the presence and function of chitin and cuticle proteins ASSCP1 and ASSCP2 in the spinning duct of silkworms. We show that chitin and these proteins are co-located in the cuticular layer of the spinning duct. Ultrastructural analysis indicates that the cuticular layer has a multilayer structure by layered stacking of the chitin laminae. After knocking down ASSCP1 and ASSCP2, the fine structure of this layer was disrupted, which had negative impacts on the mechanical properties of silk. This work clarifies the function of chitin in the spinning duct of silk-spinning arthropods. Chitin and cuticle proteins are the main components of the hard and rigid cuticular layer, providing the shearing stress during silk fibrillogenesis and regulating the final mechanical properties of silk.

Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a model of RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) N{varepsilon}-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro and in vivo, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and potentially protected their neonatal offspring from lethal challenge similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

CRISPR/Cas9-mediated gene editing provides an effective method for deciphering the molecular mechanisms underlying mosquito development and mosquito-borne disease transmission, as well as for exploring genetic control strategies. However, delivering the Cas9 ribonucleoprotein complex by embryo injection to produce genetic modifications is challenging, is mostly confined to model mosquitoes and specialized laboratories, and has low editing efficiency. Here, we established an effective Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control method, enabling the introduction of heritable mutations into Anopheles sinensis, the major malaria vector in China and Southeast Asia, via the injection of female adult mosquitoes. Injection of a mixture of P2C-DsRed and saponin resulted in red fluorescence in the ovaries, with a 100% success rate. Using this system, we knocked-out the pigment synthesis genes, Aswhite and Asyellow, using injected wild-type (WT) females mated with WT males, resulting in the highest efficiency of gene editing among mosquitoes under the same mating conditions. Furthermore, the gene-editing efficiency was increased by at least 2.1-fold using injected WT females mated with mutant males. This improved ReMOT control method exhibits high editing efficiency, with important benefits in terms of functional genomics research and genetic control strategies in An. sinensis. Moreover, this represents a convenient method for gene manipulation in laboratories that are unable to perform embryo injection or that lack embryo-injection equipment.

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and zika, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and eliminate mosquitoes in multigenerational population cages. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

Programmable site-specific nucleases have revolutionized the genome editing. However, these systems still face challenges such as guide dependency, delivery issues, and off-target effects. Harnessing the natural functions of structure-guided nucleases offer promising alternatives for generating site-specific double-strand DNA breaks. Yet, structure-guided nucleases require precise reaction conditions and validation for in-vivo applicability. To address these limitations, we developed the PNA-Coupled FokI-(d)RusA (PC-FIRA) system. PC-FIRA combines the sequence-specific binding ability of peptide nucleic acids (PNAs) with the catalytic efficiency of FokI nuclease fused to a structurally-guided inactive RusA resolvase (FokI-(d)RusA). This system allows for precise double-strand DNA breaks without the constraints of existing site-specific nuclease and structure-guided nucleases. Through in vitro optimizations, we achieved high target specificity and cleavage efficiency. This included adjusting incubation temperature, buffer composition, ion concentration, and cleavage timing. Diverse DNA structures, such as Holliday Junctions, linear, and circular DNA, were tested demonstrating the potential activity on different target forms. Further investigation has revealed the PC-FIRA system capacity for facilitating the precise deletion of large DNA fragments. This can be useful in cloning, large-fragment DNA assembly, and genome engineering, with promising applications in biotechnology, medicine, agriculture, and synthetic biology.

Synthetic circular RNA (circRNA) shows great potential in biomedical research and therapies, but impurities introduced during its synthesis can undermine circRNA efficacy. This study investigated the immunogenic potential of byproducts generated during its production. We found that trace amounts of double-stranded RNA in high molecular weight impurities, 5 triphosphates of uncircularized RNA or small intron fragments, and hydrolyzed RNA fragments significantly impact the functionality of circRNA by activating innate immune responses through the sensory molecules involved in RNA sensing. To address this, we developed a novel multi-step purification process that employs enzymatic treatments and cellulose-based filtration to selectively remove these detrimental contaminants. This tailored approach minimizes cellular immune reactions and substantially improves circRNA yields with up to more than 10 times increase. Our findings underscore the critical impact of precise contaminant management in enhancing the expression and potential therapeutic utility of circRNA. This suggests a new direction for optimizing their production for both research and clinical applications.

One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. However, all current gene drives employ a Cas9 nuclease that is constitutively active, impeding our control over their propagation abilities and limiting the generation of novel gene drive arrangements. Yet, other nucleases such as the temperature-sensitive Cas12a have not been explored for gene drive designs. To address this, we herein present a proof-of-concept gene-drive system driven by Cas12a that can be regulated via temperature modulation. Furthermore, we combined Cas9 and Cas12a to build double gene drives capable of simultaneously spreading two independent engineered alleles. The development of Cas12a-mediated gene drives provides an innovative option for designing next-generation vector control strategies to combat disease vectors and agricultural pests.

The limited thermostability and storage-induced inactivation of Moloney murine leukemia virus reverse transcriptase (MMLV RT) have constrained its applications. In this study, a high-stability mutant, MMLV RT-SV, was generated through a multi-site mutagenesis strategy targeting the nucleic acid-binding pocket. Through de novo protein design, three highly negatively charged binders with affinities of 45 nM, 362 nM, and 829 nM were developed to specifically target the positively charged nucleic acid-binding region on the surface of MMLV RT-SV. Experimental results demonstrated that these binders formed complexes with the enzyme via electrostatic interaction, significantly enhancing the thermostability and long-term storage stability of MMLV RT-SV while not affecting its RNA- dependent DNA polymerase function. This work pioneers the rational design of negatively charged binders targeting strongly positively charged nucleic acid-binding sites, overcoming the traditional stability-activity trade-off inherent in site-directed mutagenesis and directed evolution. The proposed strategy not only provides an innovative solution to address the thermal sensitivity and storage instability of reverse transcriptase but also establishes a novel paradigm for De Novo-based precision engineering of enzyme functions, demonstrating significant potential in molecular diagnostics, gene editing, and related fields.

Transferring DNA into cells to regulate cell function is a novel research field in recent decades. Chitosan is a gene vector with the properties of low-cost and safe, but high efficient delivery has remained challenging. We developed a strategy termed EEIH for endosomal explosion induced by hypertonicity, in which short-time exposure to hypertonic solutions triggers endosomes destabilization like explosions. EEIH can force chitosan/DNA polyplexes to break through the endosomal barriers to approach the nucleus, which results in boosting the transfection efficiency of chitosan in several cell lines. We demonstrate that EEIH is a significant and practical strategy in chitosan transfection system without sophisticated modification of chitosan.