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Enhanced gene transfection and induction of apoptosis in melanoma cells by branched poly(β-amino ester)s with uniformly distributed branching units

Melanoma, one of the most devastating forms of skin cancer, currently lacks effective clinical treatments. Delivery of functional genes to modulate specific protein expression to induce melanoma cell apoptosis could be a promising therapeutic approach. However, transfecting melanoma cells using non-viral methods, particularly with cationic polymers, presents significant challenges. In this study, we synthesized three branched poly(β-amino ester)s (HPAEs) with evenly distributed branching units but varying space lengths through a two-step “oligomer combination” strategy. The unique topological structure enables HPAEs to condense DNA to form nano-sized polyplexes with favorable physiochemical properties. Notably, HPAEs, especially HPAE-2 with intermediate branching unit space length, demonstrated significantly higher gene transfection efficiency than the leading commercial gene transfection reagent, jetPRIME, in human melanoma cells. Furthermore, HPAE-2 efficiently delivered the Bax-encoding plasmid into melanoma cells, leading to a pronounced pro-apoptotic effect without causing noticeable cytotoxicity. This study establishes a potent non-viral platform for gene transfection of melanoma cells by harnessing the distribution of branching units, paving the way for potential clinical applications of gene therapy in melanoma treatment.

Selenium-linked cyanobiphenyl-based liquid crystal dimers: the effects of chalcogen linkage and spacer length on the twist-bend nematic phase

ABSTRACT We synthesised selenium-linked liquid crystal dimers and examined their phase behaviour to determine the effects of chalcogen linkage and spacer length on the formation of a twist-bend nematic (NTB) phase. Two new homologous series of nonsymmetric selenoether- and ether- or thioether-linked cyanobiphenyl-based dimers (CBSenOCB and CBSenSCB, respectively) comprising an alkyl spacer chain with a carbon number (n) of 3, 5, 6, 7, 9, and 11 were developed. We also synthesised n = 6 and 11 of previously reported symmetrically selenoether-linked CBSenSeCB homologues. Conventional nematic (N) and NTB phases were observed for nearly all odd-n CBSenSeCB, CBSenOCB, and CBSenSCB dimers. The phase transition behaviours of the three homologous series were compared with those of the known symmetric ether-linked CBOnOCB, thioether-linked CBSnSCB, and asymmetric thioether- and ether-linked CBSnOCB dimers. Replacing ether with thioether or selenoether resulted in lower LC phase-transition temperatures, particularly for selenoether. Single-crystal structural analyses revealed that CBS3SCB and CBSe3SeCB exhibited folded U-shaped molecular shapes that were bent more than the V-shapes of CBOnOCB (n = 3 and 5) and CBS5SCB. In addition, thioether and selenoether linkages rendered the molecular forms of odd-spacer dimers in the crystals more bent.

The chemoenzymatic synthesis of glycan-terminated oligo(Leu)x

Introduction: Glycopeptides contain carbohydrate moieties (glycans) covalently attached to the side chain and/or terminal peptide units. Since glycans are present on cell surfaces, these constructs can potentially address a wide array of therapeutic functions. To overcome the deficiencies associated with current synthetic routes to glycopeptides, such as costly processes and toxic reagents, this work aimed to develop versatile environmentally friendly protease-catalyzed peptide synthesis routes to peptides decorated with a glycan at their N-terminus.Methods: “Grafters” were first synthesized that consist of a glycan conjugated directly, or through a spacer, to the amine group of L-Phe-ethyl ester (Phe-OEt). The role of Phe-OEt is to increase the conjugate’s recognition by the protease (papain) catalytic active site. A series of grafters were synthesized with variation of the glycan structure, linkage-chemistry, and presence of an oligo (ethylene glycol) “spacer” of varied length between the glycan and Phe-OEt moiety. High grafter efficiency will result by the successful acceptance of the grafter at the enzymes S1/S2 subsites, formation of an acyl enzyme complex and subsequent conversion to glycan-terminated oligo(Leu)x (x ≥ 1), as opposed to construction of non-glycan N-terminated oligo(Leu)x.Results and discussion: While glycan-Phe-OEt grafters without a spacer between the glycan and Phe-OEt resulted in low grafter efficiency (8.3% ± 2.0%), insertion of a short oligo (ethylene glycol) spacer between the glycan and Phe-OEt moieties (glycan-PEGn-Phe-OEt, n ≥ 3) increased the grafter efficiency by 3-fold–24.5% ± 1.8%. In addition, computational modeling was performed using Rosetta software provided insights on a molecular level of how grafter efficiency is influenced by the PEG spacer length.

Design and analysis of gate all around stacked nanosheet-DRAM for future technology node

This paper reports a stacked Dynamic random-access transistor (DRAM) memory structure that is based on gate all-around nanosheet access transistor. A TCAD study is done to compare nanosheet DRAM and conventional saddle fin recessed channel access transistor (SRCAT) in terms of DRAM electrical characteristics and its row hammer-induced leakage. The nanosheet DRAM shows superior characteristics in terms of current driving capability, speed, and refresh than SRCAT. The nanosheet DRAM also shows significantly lower hammer-induced failure as compared to SRCAT because the original leakage path from the cell to the neighboring cell gets blocked due to the nanosheet device structure. We also investigate the effect of spacer length on nanosheet DRAM characteristics and show that extended spacer length is favorable for having better DRAM characteristics due to the floating body effect. Our study demonstrates the potential, and advantages of nanosheet DRAM architecture compared to the conventional SRCAT DRAM.

Old Acquaintances and Novel Complex Structures for the Ni(II) and Cu(II) Complexes of bis-Chelate Oxime-Amide Ligands.

In the process of systematically studying the methylhydroxyiminoethaneamide bis-chelate ligands with polymethylene spacers of different lengths, L1-L3, and their transition metal complexes, a number of new Ni(II) and Cu(II) species have been isolated, and their molecular and crystal structures were determined using single-crystal X-ray diffraction. In all of these compounds, the divalent metal is coordinated by the ligand donor atoms in a square-planar arrangement. In addition, a serendipitously discovered new type of neutral Ni(II) complex, where the propane spacer of ligand L2 underwent oxidation to the propene spacer, and one of the amide groups was oxidised to the ketoimine, is also reported. The resulting ligand L2' affords the formation of neutral planar Ni(II) complexes, which are assembled in the solid state on top of each other, and yield two polymorphic structures. In both structures, the resulting infinite, exclusively parallel metal ion columns in ligand insulation may serve as precursor materials for sub-nano-conducting connectors. Overall, this paper reports the synthesis and characterisation of seven new anionic, cationic, and neutral Ni(II) and Cu(II) complexes, their crystal structures, as well as experimental and computed UV-Vis absorption spectra for two structurally similar Ni(II) complexes, yellow and red.

F16 Hybrids Derived from Steviol or Isosteviol Are Accumulated in the Mitochondria of Tumor Cells and Overcome Drug Resistance.

Steviol and isosteviol were prepared from the commercially available sweetener stevioside and converted into lipophilic F16 hybrids. Their cytotoxicity was determined in SRB assays and showed to depend on both the substitution pattern of the aromatic substituent as well as on the spacer length. Therefore, compound 25 held an IC50 (A2780) of 180 nM, thus surpassing the activity of comparable rhodamine hybrids. Several of the compounds were also able to overcome drug resistance in the A2780/A2780cis model. Extra staining experiments showed a similar subcellular accumulation pattern of the F16 hybrids as a well-established mitocan, hence proving preferential mitochondrial accumulation but also some other accumulation in other cellular areas.

Rational Approach to Improve Detergent Efficacy for Membrane Protein Stabilization.

Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy. In this study, we prepared two sets of tandem malonate-derived glucoside (TMG) variants, both of which were designed to increase the alkyl chain density in micelle interiors. The alkyl chain density was modulated either by reducing the spacer length (TMG-Ms) or by introducing an additional alkyl chain between the two alkyl chains of the original TMGs (TMG-Ps). When evaluated with a few membrane proteins including a G protein-coupled receptor, TMG-P10,8 was found to be substantially more efficient at extracting membrane proteins and also effective at preserving protein integrity in the long term compared to the previously described TMG-A13. This result reveals that inserting an additional alkyl chain between the two existing alkyl chains is an effective way to optimize detergent properties for membrane protein study. This new biochemical tool and the design principle described have the potential to facilitate membrane protein structure determination.

A self-healing waterborne acrylic latex coating based on intrinsic hydrogen bonding

Acrylic coatings suffer damage in the form of cracking, which degrades both their protective and aesthetic performance over time. Self-healing technology offers the ability to solve this problem by allowing cracks to spontaneously heal without external diagnosis or intervention, offsetting the enormous costs associated with coating damage and repair. However, there is currently no efficient self-healing acrylic coating design, and research in the area remains noticeably sparse. In this research we sought to imbue a mechanically tough methyl methacrylate (MMA)/butyl acrylate (BA)/acrylic acid (AA) acrylic coating with self-healing functionality by incorporating self-healing monomers within the formulation. We synthesized a library of four acrylic monomers containing both a long amphiphilic spacer of variable length, and the 2-ureido-4[1H]-pyrimidinone (UPy) unit, which forms strong self-complementary quadruple hydrogen bonds. These UPy-monomers were able to participate in the emulsion polymerization of MMA, BA and AA, forming intrinsic hydrogen bonding networks within the subsequent acrylic coatings. These UPy functionalized coatings displayed optical self-healing and strain recovery over 24 h both at room temperature (∼28 %), and at elevated temperatures up to 50 °C (∼80 %). The coatings also displayed repeatable self-healing after four healing cycles, relative to an MMA/BA/AA coating.

Combining Experimental and Computational Methods to Produce Conjugates of Anticholinesterase and Antioxidant Pharmacophores with Linker Chemistries Affecting Biological Activities Related to Treatment of Alzheimer's Disease.

Effective therapeutics for Alzheimer's disease (AD) are in great demand worldwide. In our previous work, we responded to this need by synthesizing novel drug candidates consisting of 4-amino-2,3-polymethylenequinolines conjugated with butylated hydroxytoluene via fixed-length alkylimine or alkylamine linkers (spacers) and studying their bioactivities pertaining to AD treatment. Here, we report significant extensions of these studies, including the use of variable-length spacers and more detailed biological characterizations. Conjugates were potent inhibitors of acetylcholinesterase (AChE, the most active was 17d IC50 15.1 ± 0.2 nM) and butyrylcholinesterase (BChE, the most active was 18d: IC50 5.96 ± 0.58 nM), with weak inhibition of off-target carboxylesterase. Conjugates with alkylamine spacers were more effective cholinesterase inhibitors than alkylimine analogs. Optimal inhibition for AChE was exhibited by cyclohexaquinoline and for BChE by cycloheptaquinoline. Increasing spacer length elevated the potency against both cholinesterases. Structure-activity relationships agreed with docking results. Mixed-type reversible AChE inhibition, dual docking to catalytic and peripheral anionic sites, and propidium iodide displacement suggested the potential of hybrids to block AChE-induced β-amyloid (Aβ) aggregation. Hybrids also exhibited the inhibition of Aβ self-aggregation in the thioflavin test; those with a hexaquinoline ring and C8 spacer were the most active. Conjugates demonstrated high antioxidant activity in ABTS and FRAP assays as well as the inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Quantum-chemical calculations explained antioxidant results. Computed ADMET profiles indicated favorable blood-brain barrier permeability, suggesting the CNS activity potential. Thus, the conjugates could be considered promising multifunctional agents for the potential treatment of AD.

A novel microchannel heat sink with staggered inlet and outlet to improve base surface temperature uniformity

Non-uniform temperature distribution can lead to performance degradation and thermal failure of electronic devices. So as to improve the base surface temperature uniformity, we proposed a novel microchannel heat sink with staggered inlet and outlet. This arrangement divides a complete flow channel into three regions, and the flow direction in each channel can be changed to form different flow modes. The effects of flow mode, the inlet and outlet spacing, Reynolds number and channel length on the temperature uniform performance of base heating surface are investigated numerically. It is found that the microchannel heat sink with staggered inlet and outlet has more uniform fluid distribution and can ensure the same highest temperature in each region to improve the base surface temperature uniformity. The optimal spacing between middle inlet and outlet decreases as Reynolds number decreases for optimal flow mode, and the improving effect of temperature uniformity becomes worse with increasing channel length. Within the calculation parameter range, compared with the straight microchannel unit, the base temperature difference and the pumping power of staggered microchannel unit with countercurrent flow in each region are reduced by 77.6% to 81.3% and 56.1% to 68.8%, respectively.

Tuning spacer length improves the functionality of the nanobody-based VEGFR2 CAR T cell

BackgroundThe chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors.ResultsWe constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells.ConclusionsThe results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment.

Oppositely-Doped Core-Shell Junctionless Nanowire FET: Design and Investigation

Junctionless Nanowire Field Effect Transistor (JL-NWFET) has garnered significant attention in recent years owing to its simplified fabrication process, achieved through uniform doping across the device. However, JL-NWFET suffers from certain drawbacks, including low drive current, insufficient volume depletion, and lateral band-to-band tunneling. To address these issues, this paper proposes Improved JL-NWFET with an oppositely doped core–shell structure along with a Dual Material gate (DMG) and high-k spacer. Furthermore, Improved JL-NWFET is optimized for parameters such as core thickness, gate oxide material, spacer material, and spacer length. The performance of Improved JL-NWFET is also evaluated in comparison with other structural variants of JL-NWFET. Notably, Improved JL-NWFET showcases an enhancement of 23.1% and 20% in ON-state current (I ON ) and transconductance (g m ), respectively when compared to the conventional JL-NWFET. Furthermore, the Subthreshold Slope (SS) experiences an 88% improvement in the Improved JL-NWFET as opposed to the JL-NWFET. Additionally, the OFF-state leakage current (I OFF ) undergoes a fivefold reduction, leading to a sixfold increase in the I ON /I OFF ratio compared to the JL-NWFET. Among all the variants of JL-NWFET devices analyzed in this paper, Improved JL-NWFET stands out with its exceptional performance characteristics.

The interplay of chromophore-spacer length in light-induced gold nanocluster self-assembly.

The light-induced self-assembly of chromophore-tethered precision nanoclusters (NCs) has recently received significant attention due to their facile control over structure, function, and reversibility under ambient conditions. However, the magnitude of assembly depends on the photoswitching efficiency, chemical structure, and proximity of the chromophore to the NC surface. Herein, using azobenzene alkyl monothiol (AMT)-capped gold NCs with two different spacer lengths (denoted as C3-NC and C9-NC), we show that reversible cis ↔ trans isomerization efficiency can be readily tuned to control the self-assembly kinetics of NCs. Irrespective of the chain length, the time required for trans-to-cis (140 s) and cis-to-trans (260 s) isomerization of individual C3-AMT and C9-AMT is identical in dichloromethane solution. When a similar experiment was performed using a solution of C3-NCs and C9-NCs, it resulted in self-assembled disc-like superstructures. Notably, the trans-to-cis photoswitching in C3-NC could reach only 65% even after 460 seconds of irradiation. On the other hand, C9-NC completed this process within 160 seconds of irradiation. The low photoswitching efficiency of the C3-NC analog is due to the short and rigid spacer length of C3-AMT ligands, which are in close proximity to the NC surface, resulting in steric hindrance experienced at the NC-chromophore interface. Importantly, the slow photoswitching in C3-NCs helps isolate and investigate the intermediates of assembly. Using high-resolution electron microscopy, atomic force microscopy, and 3D reconstruction, we show that the discs are made up of densely packed arrays of NCs. The prolonged illumination of C9-NCs results in a chain-like assembly due to the dipolar attraction between the previously assembled superstructures. The efficient photoisomerization of chromophores located away from the nanocluster surface has been identified as the key element to speed up the light-induced assembly in chromophore-tethered nanoclusters. Such information will be useful while developing nanoscale photoswitches for electrochemistry, biosensors, and electronic devices.

Optimization of Sidewall Spacer Engineering at Sub-5 nm Technology Node For JL-Nanowire FET: Digital/Analog/RF/Circuit Perspective

This manuscript presents a performance analysis of 3-stack JL-NWFETs with different spacer materials and spacer lengths. The DC and analog/RF performance is analysed at the device level, and circuit level. In single-k spacer analysis, TiO2 exhibits lowest IOFF of ∼89.28%, and largest ION/IOFF ratio with better subthreshold performance of ∼42.51% as compared to Air spacer at Lext = 7 nm. In addition, TiO2 spacer is suitable for analog applications while Air spacer for RF applications. The dual-k spacer analysis is also performed and the TiO2+Air spacer showed prodigious DC/Analog/RF performances dominating all other combinations. Further investigations into inner high-k spacer analysis (Lsp,hk) revealed that higher Lsp,hk is suitable for DC and Analog applications whereas lower Lsp,hk for RF applications. The CS amplifier designed for configurations of Lsp,hk showed better gain for higher Lsp,hk with the amplification gain of ∼4.8 V/V. Overall, this analysis serves as a beacon, guiding the future of JL-NWFET design for spellbinding nano-electronic devices at sub-5nm technology node.

Unveiling the role of linear alkyl organic cations in 2D layered tin halide perovskite field-effect transistors.

Two-dimensional (2D) tin halide perovskites are promising semiconductors for field-effect transistors (FETs) owing to their fascinating electronic properties. However, the correlation between the chemical nature of organic cations and charge carrier transport is still far from understanding. In this study, the influence of chain length of linear alkyl ammonium cations on film morphology, crystallinity, and charge transport in 2D tin halide perovskites is investigated. The carbon chain lengths of the organic spacers vary from propylammonium to heptanammonium. The increase of alkyl chain length leads to enhanced local charge carrier transport in the perovskite film with mobilities of up to 8 cm2 V-1 s-1, as confirmed by optical-pump terahertz spectroscopy. A similar improved macroscopic charge transport is also observed in FETs, only to the chain length of HA, due to the synergistic enhancement of film morphology and molecular organization. While the mobility increases with the temperature rise from 100 K to 200 K due to the thermally activated transport mechanism, the device performance decreases in the temperature range of 200 K to 295 K because of ion migration. These results provide guidelines on rational design principles of organic spacer cations for 2D tin halide perovskites and contribute to other optoelectronic applications.

Supramolecular polymerization and bulk properties relationship in ester-functionalized N-annulated perylenediimides.

The synthesis of a series of N-annulated perylenediimides (NPDIs) 1-4 with an ester group and an alkyl spacer of different length in the peripheral chains was carried out, and the influence of the side chain architecture on the self-assembly, both in solution and in the solid state, was investigated. Solution studies evidenced that the carbonyl group plays a key role in the supramolecular organization of these derivatives, changing from an H-type isodesmic polymerization (4) to a J-type cooperative process as the spacer length decreases (1-3). On the other hand, bulk assays revealed an odd-even effect that correlates with the length of the alkyl spacer. Whereas the odd-spaced derivatives (2 and 4) organize in a disordered columnar hexagonal fashion, the even-spaced ones (1 and 3) show the formation of multiple crystalline (and liquid crystalline) structures. The results presented herein highlight the importance of side chain functionalization in the design of building blocks for in-solution and bulk purposes.

Nonwoven fabric sheet with back air space serving as Helmholtz resonator

The sound absorption effect of a Helmholtz resonator was induced by creating an aperture in a nonwoven fabric sheet with a back air space, thus obtaining a sufficient sound absorption effect in a broad frequency range. In this study, transfer matrices were used to obtain the sound absorption coefficient. Transfer matrices were used to represent the following mathematically: a nonwoven fabric model based on the simplified limp-frame model, an acoustic element that considers the vibration of the nonwoven fabric sheet, the air space behind the nonwoven fabric sheet, and the aperture of the nonwoven fabric sheet. Equivalent circuits combining these transfer matrices were used to obtain sound absorption coefficient. The Helmholtz resonator sound absorption effect was more pronounced for the nonwoven fabric sheets with greater ventilation resistance. The results showed consistency between the experimental and theoretical trends. As for the sound absorption coefficient derived using the theoretical models for nonwoven fabric sheets, the results of the simplified limp-frame model were the closest to the experimental values. As the length of the back air space increased, the peak sound absorption value shifted toward low frequencies due to the sound absorption principles of both the nonwoven fabric sheet with a back air space and the Helmholtz resonator.

Physical Exercise Alters Egress Pathways for Intrinsic CSF Outflow: An Investigation Performed with Spin-labeling MR Imaging.

Cerebrospinal fluid (CSF) clearance is essential for maintaining a healthy brain and cognition by removal of metabolic waste from the central nervous system. Physical exercise has been shown to improve human health; however, the effect of physical exercise on intrinsic CSF outflow in humans remains unexplored. The purpose of this study was to investigate intrinsic CSF outflow pathways and quantitative metrics of healthy individuals with active and sedentary lifestyles. In addition, the effect of exercise was investigated among the sedentary subjects before and after 3 weeks of physical activity. This study was performed on 18 healthy adults with informed consent, using a clinical 3-Tesla MRI scanner. We classified participants into two groups based on reported time spent sitting per day (active group: < 7 hours sitting per day and sedentary group: ≥ 7 hours sitting per day). To elucidate the effect of exercise, sedentary individuals increased their activity to 3.5 hours for 3 weeks. We show that there are two intrinsic CSF egress pathways of the dura mater and lower parasagittal dura (PSD). The adults with an active lifestyle had greater intrinsic CSF outflow metrics than adults with a more sedentary lifestyle. However, after increased physical activity, the sedentary group showed improved CSF outflow metrics. This improvement was particularly notable at the lower PSD, where outflow metrics were highest among the active group. Our findings describe the relationship between physical activity and intrinsic CSF outflow and show a potential selective outflow pathway with increasing physical activity in the lower PSD pathway, potentially from the perivascular space or cortical venous subpial space.

Parametric study and optimization of cast Al-Si alloy heat exchanger using Taguchi method

A 3-D numerical model of staggered pin fin array in cast Al-Si alloy heat ex-changer was established. For further structural optimization, seven geometric parameters were studied by Taguchi method, including diameter, width, transverse spacing, longitudinal spacing, offset distance, slit length, and row number. The 32 cases with different geometric parameters were simulated numerically. The heat transfer characteristics, flow resistance characteristics, and overall performance were fully discussed. The diameter and transverse spacing have important influence on heat transfer characteristics, and the structural parameters that affect the resistance characteristics are diameter, longitudinal spacing and slit length. Then analysis of variance shows that diameter and transverse spacing have greater effects on the overall performance than other five parameters. At last, the optimized structure of staggered pin fin array was obtained, and j/f1/3 increased by 1% to 3% for volume flow rate in the range from 0.03 to 0.11 m3/s.

Unleashing the potential: type I CRISPR-Cas systems in actinomycetes for genome editing.

Covering: up to the end of 2023Type I CRISPR-Cas systems are widely distributed, found in over 40% of bacteria and 80% of archaea. Among genome-sequenced actinomycetes (particularly Streptomyces spp.), 45.54% possess type I CRISPR-Cas systems. In comparison to widely used CRISPR systems like Cas9 or Cas12a, these endogenous CRISPR-Cas systems have significant advantages, including better compatibility, wide distribution, and ease of operation (since no exogenous Cas gene delivery is needed). Furthermore, type I CRISPR-Cas systems can simultaneously edit and regulate genes by adjusting the crRNA spacer length. Meanwhile, most actinomycetes are recalcitrant to genetic manipulation, hindering the discovery and engineering of natural products (NPs). The endogenous type I CRISPR-Cas systems in actinomycetes may offer a promising alternative to overcome these barriers. This review summarizes the challenges and recent advances in CRISPR-based genome engineering technologies for actinomycetes. It also presents and discusses how to establish and develop genome editing tools based on type I CRISPR-Cas systems in actinomycetes, with the aim of their future application in gene editing and the discovery of NPs in actinomycetes.