A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0.

The zebrafish model has emerged as a reference tool for phenotypic drug screening. An increasing number of molecules have been brought from bench to bedside thanks to zebrafish-based assays over the last decade. The high homology between the zebrafish and the human genomes facilitates the generation of zebrafish lines carrying loss-of-function mutations in disease-relevant genes; nonetheless, even using this alternative model, the establishment of isogenic mutant lines requires a long generation time and an elevated number of animals. In this study, we developed a zebrafish-based high-throughput platform for the generation of F0 knock-out (KO) models and the screening of neuroactive compounds. We show that the simultaneous inactivation of a reporter gene (tyrosinase) and a second gene of interest allows the phenotypic selection of F0 somatic mutants (crispants) carrying the highest rates of mutations in both loci. As a proof of principle, we targeted genes associated with neurodevelopmental disorders and we efficiently generated de facto F0 mutants in seven genes involved in childhood epilepsy. We employed a high-throughput multiparametric behavioral analysis to characterize the response of these KO models to an epileptogenic stimulus, making it possible to employ kinematic parameters to identify seizure-like events. The combination of these co-injection, screening and phenotyping methods allowed us to generate crispants recapitulating epilepsy features and to test the efficacy of compounds already during the first days post fertilization. Since the strategy can be applied to a wide range of indications, this study paves the ground for high-throughput drug discovery and promotes the use of zebrafish in personalized medicine and neurotoxicity assessment.

Construction of luciferase-expressing Neospora caninum and drug screening.

BACKGROUND: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. METHODS: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. RESULTS: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 µM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. CONCLUSIONS: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

Contribution of elovl5a to Docosahexaenoic Acid (DHA) Synthesis at the Transcriptional Regulation Level in Common Carp, Cyprinus carpio.

Docosahexaenoic acid (DHA) is an essential nutrient for humans and plays a critical role in human development and health. Freshwater fish, such as the common carp (Cyprinus carpio), have a certain degree of DHA biosynthesis ability and could be a supplemental source of human DHA needs. The elongase of very-long-chain fatty acid 5 (Elovl5) is an important enzyme affecting polyunsaturated fatty acid (PUFA) biosynthesis. However, the function and regulatory mechanism of the elovl5 gene related to DHA synthesis in freshwater fish is not clear yet. Previous studies have found that there are two copies of the elovl5 gene, elovl5a and elovl5b, which have different functions. Our research group found significant DHA content differences among individuals in Yellow River carp (Cyprinus carpio var.), and four candidate genes were found to be related to DHA synthesis through screening. In this study, the expression level of elovl5a is decreased in the high-DHA group compared to the low-DHA group, which indicated the down-regulation of elovl5a in the DHA synthesis pathways of Yellow River carp. In addition, using a dual-luciferase reporter gene assay, we found that by targeting the 3'UTR region of elovl5a, miR-26a-5p could regulate DHA synthesis in common carp. After CRISPR/Cas9 disruption of elovl5a, the DHA content in the disrupted group was significantly higher than in the wildtype group; meanwhile, the expression level of elovl5a in the disrupted group was significantly reduced compared with the wildtype group. These results suggest that elovl5a may be down-regulating DHA synthesis in Yellow River carp. This study could provide useful information for future research on the genes and pathways that affect DHA synthesis.

Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy.

Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.

Evolving therapeutic interventions for the management and treatment of Alzheimer's disease.

Alzheimer's Disease (AD) patients experience diverse symptoms, including memory loss, cognitive impairment, behavioral abnormalities, mood changes, and mental issues. The fundamental objective of this review is to discuss novel therapeutic approaches, with special emphasis on recently approved marketed formulations for the treatment of AD, especially Aducanumab, the first FDA approved moiety that surpasses the blood-brain barrier (BBB) and reduces amyloid plaques in the brain, thereby reducing associated cognitive decline. However, it is still in the phase IV trial and is to be completed by 2030. Other drugs such as lecanemab are also under clinical trial and has recently been approved by the FDA and is also discussed here. In this review, we also focus on active and passive immunotherapy for AD as well as several vaccines, such as amyloid-beta epitope-based vaccines, amyloid-beta DNA vaccines, and stem cell therapy for AD, which are in clinical trials. Furthermore, ongoing pre-clinical trials associated with AD and other novel strategies such as curcumin-loaded nanoparticles, Crispr/ cas9, precision medicine, as well as some emerging therapies like anti-sense therapy are also highlighted. Additionally, we discuss some off-labeled drugs like non-steroidal anti-inflammatory drugs (NSAID), anti-diabetic drugs, and lithium, which can manage symptoms of AD and different non-pharmacological approaches are also covered which can help to manage AD. In summary, we have tried to cover all the therapeutic interventions which are available for the treatment and management of AD under sections approved, clinical phase, pre-clinical phase or futuristic interventions, off-labelled drugs, and non-pharmacological interventions for AD, offering positive findings and well as challenges that remain.

Gesicles packaging dCas9-VPR ribonucleoprotein complexes can combine with vorinostat and promote HIV proviral transcription.

Despite the success of combination antiretroviral therapy (cART) in HIV treatment, a cure for HIV remains elusive. Scientists postulate that HIV latent reservoirs may be a vital target in curative strategies. Vorinostat is a latency-reversing agent that has demonstrated some effectiveness in reactivating latent HIV, but complementary therapies may be essential to enhance its efficacy. One such approach may utilize the CRISPR-Cas9 system, which has evolved to include transcriptional activators such as dCas9-VPR. In this study, we explored the effects of combining vorinostat coupled with gesicle-mediated delivery of dCas9-VPR in promoting the transcription of integrated HIV proviruses in HIV-NanoLuc CHME-5 microglia and J-Lat 10.6 lymphocytes. We confirmed that dCas9-VPR ribonucleoprotein complexes can be packaged into gesicles and application to cells successfully induced HIV transcription through interactions with the HIV LTR. Vorinostat also induced significant increases in proviral transcription but generated inhibition of cellular proliferation (microglia) or cell viability (lymphocytes) starting at 1,000 nM and higher concentrations. Experiments combining dCas9-VPR gesicles and vorinostat confirmed the enhanced transcriptional activation of the HIV provirus in microglia but not lymphocytes. Thus, a combination of dCas9-VPR gesicles with other latency-reversing agents may provide a complementary method to activate latent HIV in future studies utilizing patient-derived cells or small animal models.

AAV-based gene editing of type 1 collagen mutation to treat osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility, low bone mass, fractures, and extraskeletal manifestations. Since OI is commonly caused by single-nucleotide mutation(s) in the COL1A1 or COL1A2 genes encoding type I collagens, we developed a genome-editing strategy to correct a Col1a2 mutation in an OIM mouse model resembling a severe dominant form of human type III OI. Using a recombinant adeno-associated virus (rAAV), we delivered CRISPR-Cas9 to bone-forming osteoblast-lineage cells in the skeleton. Homology-directed repair (HDR)-mediated gene editing efficiency in these cells was improved when CRISPR-Cas9 was coupled with a donor AAV vector containing a promoterless partial mouse Col1a2 complementary DNA sequence. This approach effectively reversed the dysregulation of osteogenic differentiation by a Col1a2 mutation in vitro. Furthermore, systemic administration of dual rAAVs in OIM mice lowered bone matrix turnover rates by reducing osteoblast and osteoclast development while improving the cellular network of mechano-sensing osteocytes embedded in the bone matrix. This strategy significantly improved bone architecture/mass/mineralization, skeletal deformities, grip strength, and spontaneous fractures. Our study is the first demonstration that HDR-mediated gene editing via AAV-mediated delivery effectively corrects a collagen mutation in OI osteoblasts and reverses skeletal phenotypes in OIM mice.

Fostering plant growth performance under drought stress using rhizospheric microbes, their gene editing, and biochar.

Stress due to drought lowers crop yield and frequently leads to a rise in food scarcity. Plants' intricate metabolic systems enable them to tolerate drought stress, but they are unable to handle it well. Adding some external, environmentally friendly supplements can boost plant growth and productivity when it comes to drought-stressed plants. In order to prevent the detrimental effects of drought in agricultural regions, environmentally friendly practices must be upheld. Plant growth-promoting rhizobacteria (PGPR) can exhibit beneficial phytostimulation, mineralization, and biocontrol activities under drought stress. The significant impact of the PGPR previously reported has not been accepted as an effective treatment to lessen drought stress. Recent studies have successfully shown that manipulating microbes can be a better option to reduce the severity of drought in plants. In this review, we demonstrate how modifying agents such as biochar, PGPR consortia, PGPR, and mycorrhizal fungi can help overcome drought stress responses in crop plants. This article also discusses CRISPR/Cas9-modifiable genes, increase plant's effectiveness in drought conditions, and increase plant resistance to drought stress. With an eco-friendly approach in mind, there is a need for practical management techniques having potential prospects based on an integrated strategy mediated by CRISPR-Cas9 editing, PGPR, which may alleviate the effects of drought stress in crops and aid in achieving the United Nation Sustainable Development Goals (UN-SDGs-2030).

Androgen receptor deficiency is associated with reduced aromatase expression in the ventromedial hypothalamus of male cichlids.

Social behaviors are regulated by sex steroid hormones, such as androgens and estrogens. However, the specific molecular and neural processes modulated by steroid hormones to generate social behaviors remain to be elucidated. We investigated whether some actions of androgen signaling in the control of social behavior may occur through the regulation of estradiol synthesis in the highly social cichlid fish, Astatotilapia burtoni. Specifically, we examined the expression of cyp19a1, a brain-specific aromatase, in the brains of male A. burtoni lacking a functional ARα gene (ar1), which was recently found to be necessary for aggression in this species. We found that cyp19a1 expression is higher in wild-type males compared to ar1 mutant males in the anterior tuberal nucleus (ATn), the putative fish homolog of the mammalian ventromedial hypothalamus, a brain region that is critical for aggression across taxa. Using in situ hybridization chain reaction, we determined that cyp19a1+ cells coexpress ar1 throughout the brain, including in the ATn. We speculate that ARα may modulate cyp19a1 expression in the ATn to govern aggression in A. burtoni. These studies provide novel insights into the hormonal mechanisms of social behavior in teleosts and lay a foundation for future functional studies.

Gpr149 is involved in energy homeostasis in the male mouse.

GPR149 is an orphan receptor about which little is known. Accordingly, in the present study, we mapped the tissue expression of Gpr149 in mice using three complementary approaches: quantitative PCR, in situ hybridization, and a newly generated Gpr149-Cre reporter mouse model. The strongest expressions of Gpr149 were observed in neurons of the islands of Calleja, the ventromedial hypothalamus, and the rostral interpeduncular nucleus. Moderate-to-low expression was also observed in the basal forebrain, striatum, hypothalamus, brainstem, and spinal cord. Some Gpr149 expression was also detected in the primary afferent neurons, enteric neurons, and pituitary endocrine cells. This expression pattern is consistent with the involvement of GPR149 signaling in the regulation of energy balance. To explore the physiological function of GPR149 in vivo, we used CRISPR-Cas9 to generate a global knockout allele with mice lacking Gpr149 exon 1. Preliminary metabolic findings indicated that Gpr149-/- mice partially resist weight gain when fed with a high-fat diet and have greater sensitivity to insulin than control mice. In summary, our data may serve as a resource for future in vivo studies on GPR149 in the context of diet-induced obesity.

CRISPR/Cas9-based genome editing of 14 lipid metabolic genes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.

Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

CRISPR/Cas9-meditated gene knockout in pigs proves that LGALS12 deficiency suppresses the proliferation and differentiation of porcine adipocytes.

LGALS12, also known as galectin12, belongs to the galectin family with ß-galactoside-binding activity. We previously reported that LGALS12 is an important regulator of adipogenesis in porcine adipocytes in vitro, but its value in pig breeding needed to be explored in vivo. In this study, we used CRISPR/Cas9 to construct porcine fetal fibroblasts (PFFs) with a 43 bp deletion in LGALS12 exon 2. Using these PFFs as donor cells, a LGALS12 knockout pig model was generated via somatic cell nuclear transfer. Primary cultures of porcine intramuscular (IM) and subcutaneous (SC) adipocytes were established using cells from LGALS12 knockout pigs and wild-type pigs. A comparison of these cells proved that LGALS12 deficiency suppresses cell proliferation via the RAS-p38MAPK pathway and promotes lipolysis via the PKA pathway in both IM and SC adipocytes. In addition, we observed AKT activation only in IM adipocytes and suppression of the Wnt/ß-catenin only in SC adipocytes. Our findings suggest that LGALS12 deficiency affects the adipogenesis of IM and SC adipocytes through different mechanisms.

Alkaline Phosphatase PhoD Mutation Induces Fatty Acid and Long-Chain Polyunsaturated Fatty Acid (LC-PUFA)-Bound Phospholipid Production in the Model Diatom Phaeodactylum tricornutum.

With rapid growth and high lipid contents, microalgae have become promising environmentally friendly candidates for renewable biodiesel and health supplements in our era of global warming and energy depletion. Various pathways have been explored to enhance algal lipid production, especially gene editing. Previously, we found that the functional loss of PhoD-type alkaline phosphatase (AP), a phosphorus-stress indicator in phytoplankton, could lead to increased lipid contents in the model diatom Phaeodactylum tricornutum, but how the AP mutation may change lipid composition remains unexplored. This study addresses the gap in the research and investigates the effects of PhoD-type AP mutation on the lipid composition and metabolic regulation in P. tricornutum using transcriptomic and lipidomic analyses. We observed significantly modified lipid composition and elevated production of fatty acids, lysophosphatidylcholine, lysophosphatidylethanolamine, ceramide, phosphatidylinositol bisphosphate, and monogalactosylmonoacylglycerol after PhoD_45757 mutation. Meanwhile, genes involved in fatty acid biosynthesis were upregulated in mutant cells. Moreover, the mutant exhibited increased contents of ω-3 long-chain polyunsaturated fatty acid (LC-PUFA)-bound phospholipids, indicating that PhoD_45757 mutation could improve the potential bioavailability of PUFAs. Our findings indicate that AP mutation could influence cellular lipid synthesis and probably redirect carbon toward lipid production and further demonstrate that AP mutation is a promising approach for the development of high-value microalgal strains for biomedical and other applications.

CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.

CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.

Salt and osmotic stress can improve the editing efficiency of CRISPR/Cas9-mediated genome editing system in potato.

CRISPR/Cas9-mediated genome editing technology has been widely used for the study of gene function in crops, but the differences between species have led to widely varying genome editing efficiencies. The present study utilized a potato hairy root genetic transformation system and incorporated a rapid assay with GFP as a screening marker. The results clearly demonstrated that salt and osmotic stress induced by NaCl (10 to 50 mM) and mannitol (50 to 200 mM) treatments significantly increased the positive rates of genetic transformation mediated by A. rhizogenes and the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato. However, it was observed that the regeneration of potato roots was partially inhibited as a result. The analysis of CRISPR/Cas9-mediated mutation types revealed that chimeras accounted for the largest proportion, ranging from 62.50% to 100%. Moreover, the application of salt and osmotic stress resulted in an increased probability of null mutations in potato. Notably, the highest rate of null mutations, reaching 37.5%, was observed at a NaCl concentration of 10 mM. Three potential off-target sites were sequenced and no off-targeting was found. In conclusion, the application of appropriate salt and osmotic stress significantly improved the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato, with no observed off-target effects. However, there was a trade-off as the regeneration of potato roots was partially inhibited. Overall, these findings present a new and convenient approach to enhance the genome editing efficiency of the CRISPR/Cas9-mediated gene editing system in potato.

Steerable Microneedles Enabling Deep Delivery of Photosensitizers and CRISPR/Cas9 Systems for Effective Combination Cancer Therapy.

Although gene therapy has shown prospects in treating triple-negative breast cancer, it is insufficient to treat such a malignant tumor. Herein, nanoparticles (NPs)-embedded dissolving microneedles (IR780-PL/pFBXO44@MNs) with steerable and flectional property were developed to achieve the codelivery of FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and hydrophobic photosensitizers. For improved NP penetration in tumor tissue, collagenase@MNs were preapplied to degrade the tumor matrix. Under light irradiation, IR780 exhibited remarkable phototherapy, while the escape efficiency of NPs from lysosomes was improved. pFBXO44 was subsequently released in tumor cell cytoplasm via reducing the disulfide bonds of NPs, which could specifically knock out the FBXO44 gene to inhibit the migration and invasion of tumor cells. As a result, tumor cells were eradicated, and lung metastasis was effectively suppressed. This micelle-incorporated microneedle platform broadens the potential of combining gene editing and photo synergistic cancer therapy.

New advances in the treatments of drug-resistant tuberculosis.

INTRODUCTION: TB is associated with high mortality and morbidity among infected individuals and a high transmission rate from person to person. Despite the availability of vaccines and several anti-TB,TB infection continues to increase. Global resistance to TB remains the greatest challenge. There has not been extensive research into a new treatment and management strategy for TB resistance therapy. This review is based on a review of new advances and alternative drugs in the treatment of drug-resistant TB. AREAS COVERED: New drug-resistant Mycobacterium tuberculosis therapy involves a combination of the latest TB drugs, new anti-TB drugs based on medicinal plant extracts for drug-resistant TB, mycobacteriophage therapy, the CRISPR/Cas9 system, and nanotechnology. EXPERT OPINION: It is necessary to determine the function of individual gene alterations in drug-resistant TB. A combination of the most recent anti-TB drugs, such as bedaquiline and delamanid, is recommended. Longitudinal studies and animal model experiments with some medicinal plant extracts are required for better results. Nanotechnology has the potential to reduce drug side effects. Useful efficacy of phage therapy and CRISPR-cas9 technology as adjunct therapies for the management of drug-resistant TB.