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Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent Trans IT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.

Background CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. Results We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis , for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis . Conclusion Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis , and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.

The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo to cells and tissues of interest remains challenging. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). This is achieved by using a microfluidic electroporation approach in which a combination of nano- and milli-second pulses produces large amounts of IFN-γ mRNA-loaded sEVs with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic sEVs (imsEV) preferentially target glioblastoma cells and generate potent antitumour activities in vivo, including against tumours intrinsically resistant to immunotherapy. Together, these results provide an adaptive approach to engineering mRNA-loaded sEVs with targeting functionality and pave the way for their adoption in cancer immunotherapy applications. There is an emerging interest in the use of mRNA therapeutics in cancer treatment, but their precise in vivo delivery remains a challenge. Here the authors develop IFN-γ mRNA-loaded small extracellular vesicles (sEVs) with CD64 overexpressed on their surface and demonstrate its efficacy in glioblastoma mouse models resistant to immunotherapy.

The past decade has seen growing interest in bacterial engineering for therapeutically relevant applications. While early efforts focused on repurposing genetically tractable model strains, such as Escherichia coli , engineering gut commensals is gaining traction owing to their innate capacity to survive and stably propagate in the intestine for an extended duration. Although limited genetic tractability has been a major roadblock, recent advances in systems and synthetic biology have unlocked our ability to effectively harness native gut commensals for therapeutic and diagnostic purposes, ranging from the rational design of synthetic microbial consortia to the construction of synthetic cells that execute “sense-and-respond” logic operations that allow real-time detection and therapeutic payload delivery in response to specific signals in the intestine. In this review, we outline the current progress and latest updates on microbial therapeutics, with particular emphasis on gut commensal engineering driven by synthetic biology and systems understanding of their molecular phenotypes. Finally, the challenges and prospects of engineering gut commensals for therapeutic applications are discussed.

Switchgrass ( Panicum virgatum ) is an excellent feedstock for biofuel production. While genetic transformation is routinely done in lowland switchgrass, upland cultivars remain recalcitrant to genetic transformation. Here we report the establishment of an efficient and reproducible transformation protocol for two upland cultivars, ‘Summer’ and ‘Blackwell’, by ectopic overexpression of morphogenic genes, Baby boom ( Bbm ) and Wuschel2 ( Wus2 ). Two auxotrophic Agrobacterium strains, LBA4404Thy- and EHA105Thy-, each harboring the same construct containing ZmBbm , ZmWus2 , and a green fluorescence protein (GFP) gene, ZsGreen1 , were used to infect immature leaf segments derived from in vitro grown seedlings. The Agrobacterium strains also contain a transformation helper plasmid that carry additional copies of Agrobacterium virulence genes. GFP-expressing calli were identified and selected for regeneration. The highest transformation efficiency of 6% was obtained for the tetraploid cultivar Summer when LBA4404Thy- was used for infection, which is twice of that for the octoploid cultivar Blackwell. LBA4404Thy- consistently outperformed EHA105Thy- on transformation frequency across the two cultivars. Fifteen randomly selected putative transgenic plants of Summer and Blackwell, representing independent callus events, were confirmed as transgenic by the presence of the transgene, ZmAls , and the absence of AtuFtsZ , a chromosomal gene specific to the Agrobacterium strain LBA4404 using polymerase chain reaction. Transgene integration and expression was further confirmed by the detection of GFP in roots, and the resistance to herbicide injury to leaves of selected putative transgenic plants. The ZmBbm and ZmWus2 genes were successfully removed from 40 to 33.3% of the transgenic plants of Summer and Blackwell, respectively, via the Cre-Lox recombination system upon heat treatment of GFP-expressing embryogenic calli. Our successful transformation of recalcitrant upland switchgrass provides a method for gene function analysis and germplasm enhancement via biotechnology.

PurposeThis paper aims to investigate how employee–organization relationship (EOR) outcomes – types and qualities – are interrelated and how employees' perceptions of types (exchange and communal EORs) and qualities (trust, satisfaction, commitment, and control mutuality) play a role in their evaluations of symmetrical internal communication (SIC) and employee job engagement (EJE).Design/methodology/approachThis study conducted an online survey of full-time employees (N = 804) from major US industries. This study performed a confirmatory factor analysis to check the validity and reliability of the measurement model using latent variables and then conducted structural equation modeling.FindingsThe findings demonstrate that employees' perceptions of both exchange and communal EORs are associated with each of the four EOR qualities. The results also show that only communal EORs have a significant relationship with perceived SIC and that employees' perceptions about one of the EOR quality indicator, satisfaction with an organization, has a significant association with their perceived EJE.Originality/valueThis study contributes to relationship management theory within the internal context by examining the interrelationship between each of the EOR types and qualities that are perceived by employees. This paper also suggests the practical importance of developing not only communal but also exchange EORs to enhance EOR quality. Additionally, the results imply that SIC programs could help to enhance employees' perceptions of communal EORs and employees could be engaged in their workplace when they are satisfied with their organizations.

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.

Hamartoma is characterized by the abnormal proliferation of cells and tissues within the originating organ. Apocrine hamartoma (AH) affecting the skin, especially in the perianal area, has not yet been reported in dogs. A 7‐year‐old neutered male Poodle presented with multiple perianal papules and pruritus. Histopathological examination revealed significant enlargement of the apocrine glands and ducts, accompanied by mononuclear cell infiltration that extended into the subcutis. Given the location, number, depth and extent of the lesion, CO2 laser ablation was chosen over surgical resection. Complete recovery was observed at the surgical site 1 month after treatment, and pruritic symptoms were markedly reduced. CO2 laser ablation is a relatively safe and effective palliative treatment for perianal AH in dogs. A 7‐year‐old Poodle dog presented with multiple perianal papules and persistent pruritis that was unresponsive to steroids and antibiotics. Diagnosis revealed apocrine hamartoma affecting the perianal region, a condition not previously reported in dogs. This case report describes the histopathologic features of perianal apocrine hamartoma and introduces CO2 laser as an effective treatment modality, aiming at alleviating the clinical signs rather than providing a definitive cure.

Abstract Background Nucleated red blood cells (nRBCs) are increased by disease processes and hematopoietic stress. Objectives To evaluate the utility of nRBCs as a marker of disease severity and prognosis in dogs with systemic inflammatory response syndrome (SIRS). Animals Sixty-two client-owned dogs met the criteria of SIRS without anemia. Methods nRBC-positive (nRBCs: ≥5/500, n = 32) and nRBC-negative (nRBCs: <5/500, n = 30) dogs were classified, and clinicopathological data, Acute Patient Physiologic and Laboratory Evaluation (APPLEfast) scores, cytokines, 2- and 4-weeks survival were compared. Results The median WBC (17.63, interquartile range [IQR]: 11.72-20.24 × 109/L), neutrophils (12.28, IQR: 7.17-16.88 × 109/L), band neutrophils (1288.5, IQR: 252.5-2575 cells/μL), serum IL-6 (731.80, IQR: 299.79-5522.05 pg/mL), and plasma C-reactive protein (4.10, IQR: 1.00-8.58 mg/L) were significantly higher in nRBC-positive dogs than negative dogs (11.27, IQR: 7.63-15.13 × 109/L; 7.57, IQR: 4.96-11.71 × 109/L; 62.5, IQR: 0-350.25 cells/μL; 232.30, IQR: 99.33-447.01 pg/mL; 0.40, IQR: 0.10-3.00 mg/L, respectively; P < .05). The median reticulocyte count (87.95, IQR: 52.45-130.55 × 103/μL) and serum IL-3 (40.94, IQR: 29.85-53.52 ng/L) were also significantly greater in nRBC-positive dogs than nRBC-negative dogs (46.00, IQR: 26.43-68.15 × 103/μL; 25.24, IQR: 21.65-37.40 ng/L, respectively; P < .01). The presence of circulating nRBCs, but not the reticulocyte count, at admission was predictive of death in dogs with SIRS at 2 weeks (P = .01, AUC: 0.729) and 4 weeks (P = .002, AUC: 0.731). The overall survival time was shorter in nRBC-positive dogs (95% CI, 47.35-113.90) than nRBC-negative dogs (95% CI, 90.92-135.55; P = .03). Conclusions and Clinical Importance Measuring peripheral nRBCs in dogs with SIRS is rapid and clinically applicable, reflecting disease severity and associated prognosis.