Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System

CRISPR （clustered regularly interspaced short palindromic repeats）/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel （insertion and deletion） mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA- Cas9 constructs were transformed into protoplasts followed by sequencmg the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM （protospacer adjacent motif） or having more than two mismatches. Together. our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.

Orange-derived extracellular vesicles nanodrugs for efficient treatment of ovarian cancer assisted by transcytosis effect

Extracellular vesicles(EVs)have recently received much attention about the application of drug carriers due to their desirable properties such as nano-size,biocompatibility,and high stability.Herein,we demonstrate orange-derived extracellular vesicles(OEV)nanodrugs(DN@OEV)by modifying cRGD-targeted doxorubicin(DOX)nanoparticles(DN)onto the surface of OEV,enabling significantly enhancing tumor accumulation and penetration,thereby efficiently inhibiting the growth of ovarian cancer.The obtained DN@OEV enabled to inducement of greater transcytosis capability in ovarian cancer cells,which presented the average above 10-fold transcytosis effect compared with individual DN.It was found that DN@OEV could trigger receptor-mediated endocytosis to promote early endosome/recycling endosomes pathway for exocytosis and simultaneously reduce degradation in the early endosomes-late endosomes-lysosome pathway,thereby inducing the enhanced transcytosis.In particular,the zombie mouse model bearing orthotopic ovarian cancer further validated DN@OEV presented high accumulation and penetration in tumor tissue by the transcytosis process.Our study indicated the strategy in enhancing transcytosis has significant implications for improving the therapeutic efficacy of thedrugdelivery system.

Characterization and of Ralstonia TAL-Like DNA-Binding Specificities Effectors

Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.

TALENs:Customizable Molecular DNA Scissors for Genome Engineering of Plants

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.

pH-sensitive and bubble-generating mesoporous silica-based nanoparticles for enhanced tumor combination therapy

Chemotherapy has been a major option in clinic treatment of malignant tumors.However,single chemotherapy faces some drawbacks,such as multidrug resistance,severe side effects,which hinder its clinic application in tumor treatment.Multifunctional nanoparticles loading with chemotherapeutic agent and photosensitizer could be a promising way to efficiently conduct tumor combination therapy.In the current study,a novel pH-sensitive and bubble-generating mesoporous silica-based drug delivery system(denoted as M(a)D@PI-PEG-RGD)was constructed.Ammonium bicarbonate(NH4 HCO3;abc)and chemotherapeutic agent doxorubicin(DOX)were loaded into the pores of mesoporous silica.Indocyanine green(ICG)as a photothermal and photodynamic agent was loaded onto the polydopamine(PDA)layer surface.The synthesized nanoparticles displayed a narrow polydispersity(PDI)and small particle size as characterized through dynamic light scattering-autosizer analysis.The nanoparticles also showed high targeting efficacy through RGD modification as indicated by cellular uptake and animal studies.DOX release analysis confirmed that the nanoparticles were pH-dependent and that NH4 HCO3 accelerated drug release.At the same time,the nanoparticles had obvious photothermal and photodynamic effects performed by ICG which restrained tumor growth remarkably.In summary,the multifunctional nanoparticles presented a promising system for combination therapy.