The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/ tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a lightresponsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.
BackgroundThe COVID-19 pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines.AimsWe hypothesised that passive immunity engineered by the in vivo expression of anti-SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored-immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age.MethodsWe developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs.ConclusionsA single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV-2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV-2 vaccines. The in vivo expression of multiple anti-SARS-CoV-2 mAbs could enhance protection and prevent rapid mutational escape.
Biofilm is an aggregate of consortium bacteria that adhere to each other on a surface. It is usually protected by the exopolysaccharide layer. Various invasive medical procedures, such as catheterization, endotracheal tube installation, and contact lens utilization, are vulnerable to biofilm infection. The National Institute of Health (NIH) estimates 65% of all microbial infections are caused by biofilm. Periplasmic α-amylase (MalS) is an enzyme that hydrolyzes α-1, 4-glicosidic bond in glycogen, starch, and others related polysaccharides in periplasmic space. Another protein called hemolysin-α (HlyA) is a secretion signal protein on C terminal of particular peptide in gram negative bacteria. We proposed a novel recombinant plasmid expressing α-amylase and hemolysin-α fusion in pSB1C3 which is cloned into E.coli to enable α-amylase excretion to extracellular for degrading biofilm polysaccharides content, as in starch agar.Microtiter assay was performed to analyze the reduction percentage of biofilm by adding recombinant E.coli into media. This system is more effective in degrading biofilm from gram positive bacteria i.e.: Bacillus substilis (30.21%) and Staphylococcus aureus (24.20%), and less effective degrading biofilm of gram negative i.e.: Vibrio cholera (5.30%), Pseudomonas aeruginosa (8.50%), Klebsiella pneumonia (6.75%) and E. coli (-0.6%). Gram positive bacteria have a thick layer of peptidoglycan, causing the enzyme to work more effectively in degrading polysaccharides. AbstrakEkspresi dan Penelitian Fungsi Protein Fusi α-Amilase dan Hemolisin-α sebagai suatu Penerapan dalam Penurunan Polisakarida Biofilm. Biofilm adalah sekumpulan bakteria yang saling melekat satu sama lain pada suatu permukaan. Biofilm ini biasanya dilindungi oleh lapisan eksopolisakarida. Berbagai prosedur medis yang pro-aktif, seperti kateterisasi, instalasi alat bantuan pernafasan, dan penggunaan lensa kontak mata, rentan terhadap infeksi biofilm. NIH (National Institute of Health -Institusi Kesehatan Nasional) memperkirakan 65% dari semua infeksi mikroba disebabkan oleh biofilm. Enzim α-amilase periplasma (MalS) merupakan suatu enzim yang menghidrolisis α-1, ikatan 4-glikosidik dalam glikogen, zat tepung, dan lainnya terkait polisakarida pada ruang periplasma. Protein lainnya yang disebut hemolisin-α (HlyA) merupakan protein sinyal sekresi pada terminal C dari peptida tertentu dalam bakteria gram-negatif. C merupakan protein sinyal sekresi pada terminal C dari peptida tertentu dalam bakteria gram-negatif. Kami mengusulkan suatu plasmid rekombinan baru mengekspresikan fusi α-amilase and hemolisin-α dalam pSB1C3 yang diklon menjadi E. coli untuk memungkinkan ekskresi α-amilase ke luar sel tubuh (ekstraselular) untuk menurunkan isi polisakarida biofilm, seperti dalam agar zat tepung. Tes dengan tabung kecil dilakukan untuk menganalisis persentase pengurangan biofilm dengan menambahkan E. coli rekombinan ke dalam media. Sistem ini lebih efektif dalam menurunkan tingkat biofilm dari bakteria gram-positif, seperti Bacillus subst...
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