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The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed., (© 2023. Controlled Release Society.)

Atractylodes lancea (Thunb.) DC. is a well-known medicinal herb in China, containing abundant active components, including a variety of sesquiterpenoids. Owing to a shortage of wild resources, artificial cultivation has become the main breeding mode, leading to the germplasm degradation. In preliminary research, our research group found that a mutant tissue culture seedling of A. lancea is an excellent germplasm resource, characterized by early stem growth and higher sesquiterpenoid content than that of the wild type. In this study, the physiological and biochemical mechanisms underlying efficient sesquiterpenoids synthesis by this mutant A. lancea were systematically evaluated. The results showed that the photosynthetic efficiency, central carbon metabolism efficiency, and energy metabolism efficiency were significantly improved in mutant A. lancea compared with the wild type, and the content of endogenous hormones, such as gibberellin and jasmonic acid, changed significantly. In addition, levels of key metabolites and the expression level of key genes in the mevalonate and 2-C-methyl-d-erythritol-4-phosphate pathways were significantly higher in mutant type than in wild type, resulting in elevated sesquiterpenoid synthesis in the mutant. These physiological and biochemical properties explain the rapid growth and high sesquiterpenoid content of mutant A. lancea ., Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-022-02240-5., Competing Interests: Conflict of interestAuthors declare that they have no conflict of interest and the publication of the work has been approved by all co-authors., (© The Author(s), under exclusive licence to Springer Nature B.V. 2022.)

Composting has become the most important way to recycle medicinal herbal residues (MHRs). The traditional composting method, adding a microbial agent at one time, has been greatly limited due to its low composting efficiency, mutual influence of microbial agents, and unstable compost products. This study was conducted to assess the effect of multi-phase inoculation on the lignocellulose degradation, enzyme activities, and fungal community during MHRs composting. The results showed that multi-phase inoculation treatment had the highest thermophilic temperature (68.2 °C) and germination index (102.68%), significantly improved available phosphorus content, humic acid, and humic substances concentration, accelerated the degradation of cellulose and lignin, and increased the activities of cellulase in the mature phase, xylanase, manganese peroxidase, and utilization of phenolic compounds. Furthermore, the non-metric multi-dimensional scaling showed that the composting process and inoculation significantly influenced fungal community composition. In multi-phase inoculation treatment, Thermomyces in mesophilic, thermophilic, and mature phase, unclassified_Sordariales, and Coprinopsis in mature phase were the dominant genus that might be the main functional groups to degrade lignocellulose and improve the MHRs composting process.

A novel isolate, designated strain KLBMP 1282(T) was isolated from the surface-sterilized leaves of a coastal halophyte Tamarix chinensis Lour., collected from Nantong, Jiangsu Province, east of China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain belongs to the genus Pseudonocardia, being most closely related to Pseudonocardia kongjuensis LM 157(T) (98.33 %), Pseudonocardia autotrophica IMSNU 20050(T) (97.77 %), Pseudonocardia endophytica YIM 56035(T) (97.63 %), Pseudonocardia ammonioxydans H9 (T) (97.62 %) and Pseudonocardia compacta IMSNU 20111(T) (97.56 %); similarity to other type strains of the genus Pseudonocardia was <97.5 %. Chemotaxonomic data confirmed the affiliation of strain KLBMP 1282(T) to the genus Pseudonocardia. Strain KLBMP 1282(T) contained MK-8(H(4)) as the predominant ubiquinone and iso-C(16:0) as the major fatty acid. The polar lipids detected in strain KLBMP 1282(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, one unknown phospholipid and four unknown glycolipids. The DNA G + C content of strain KLBMP 1282(T) was 73.1 mol %. The results of DNA-DNA hybridizations and the phylogenetic analysis, together with the phenotypic and biochemical tests, allowed the differentiation of strain KLBMP 1282(T) from strains of other recognized Pseudonocardia species. Therefore, strain KLBMP 1282(T) represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia nantongensis sp. nov. is proposed. The type strain is KLBMP 1282(T) (=KCTC 29053(T) = NBRC 108677(T)).

A novel Gram-positive, motile, non-spore-forming coccus-shaped bacterial strain, designated KLBMP 1274(T), was isolated from a halophytic plant (Limonium sinense) collected from the coastal region of Nantong, Jiangsu Province, in east China. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain KLBMP 1274(T) belongs to the genus Kineococcus and is closely related to Kineococcus rhizosphaerae RP-B16(T) (98.72 %), Kineococcus aurantiacus IFO 15268(T) (98.71 %), Kineococcus radiotolerans SRS30216(T) (98.69 %) and Kineococcus gynurae KKD096(T) (97.33 %). The 16S rRNA gene sequence similarity to other species of the genus Kineococcus was <97 %. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid, with arabinose and galactose as the characteristic sugars. The predominant menaquinone was MK-9(H(2)). The polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, an unknown phospholipid, an unknown glycolipid, and three unknown lipids. Major cellular fatty acids were found to be anteiso-C(15: 0) and iso-C(14: 0). The chemotaxonomic data for strain KLBMP 1274(T) were typical of the genus Kineococcus. The total DNA G+C content was 73.4 mol %. DNA-DNA relatedness and differential phenotypic data demonstrated that strain KLBMP 1274(T) was clearly distinguished from all closely related species of the genus Kineococcus. Thus, strain KLBMP 1274(T) represents a novel species of the genus Kineococcus, for which the name Kineococcus endophytica sp. nov. is proposed. The type strain is KLBMP 1274(T) (=KCTC 19886 (T) = NBRC 108674(T)).

The effects of the endophytic fungus Gilmaniella sp. and its elicitor on the defense and metabolic responses of host plants Atractylodes lancea were investigated, in order to understand how to utilize endophytic fungi and their elicitor resources better. The results showed that the promotion effect of the fungus on the growth of host plantlets was much better than that of its elicitor. Both fungus and elicitor enhanced defense-related enzyme activities. In fungus-inoculated groups, phenylalanine ammonia lyase and polyphenol oxidase activities increased slowly, and reached a maximum level during the later stages, whereas peroxidase activity peaked in the first few days. Additionally, the activities of chitinase and β-1,3-glucanase were significantly higher than those of the control plants. In elicitor-treated groups, however, most of the enzymes were activated during the early stage, and their highest levels were generally lower than those of the fungus-inoculated groups. Compared with the elicitor, fungal infection improved the photosynthetic rate of the host, and increased carbohydrate levels as well as chlorophyll content in host leaves. The total content of the four main components of volatile oil was also increased in elicitor-treated groups, but there was no particular pattern in this increase. Meanwhile, in the fungus-inoculated groups, the content of atractylone significantly increased with time, while the content of β-eudesmol decreased. These results indicated that fungal elicitor could substantially improve the total content of volatile oil, while the fungus could more effectively enhance the quality of herbal medicines.