Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.

BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies. RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

Designing hybrid CRISPR-Cas12 and LAMP detection systems for treatment-resistant Plasmodium falciparum with in silico method.

Genes associated with drug resistance of first line drugs for Plasmodium falciparum have been identified and characterized of which three genes most commonly associated with drug resistance are P. falciparum chloroquine resistance transporter gene (PfCRT), P. falciparum multidrug drug resistance gene 1 (PfMDR1), and P. falciparum Kelch protein K13 gene (PfKelch13). Polymorphism in these genes could be used as molecular markers for identifying drug resistant strains. Nucleic acid amplification test (NAAT) along with DNA sequencing is a powerful diagnostic tool that could identify these polymorphisms. However, current NAAT and DNA sequencing technologies require specific instruments which might limit its application in rural areas. More recently, a combination of isothermal amplification and CRISPR detection system showed promising results in detecting mutations at a nucleic acid level. Moreover, the Loop-mediated isothermal amplification (LAMP)-CRISPR systems offer robust and straightforward detection, enabling it to be deployed in rural and remote areas. The aim of this study was to develop a novel diagnostic method, based on LAMP of targeted genes, that would enable the identification of drug-resistant P. falciparum strains. The methods were centered on sequence analysis of P. falciparum genome, LAMP primers design, and CRISPR target prediction. Our designed primers are satisfactory for identifying polymorphism associated with drug resistant in PfCRT, PfMDR1, and PfKelch13. Overall, the developed system is promising to be used as a detection method for P. falciparum treatment-resistant strains. However, optimization and further validation the developed CRISPR-LAMP assay are needed to ensure its accuracy, reliability, and feasibility.

Immunoinformatics Identification of the Conserved and Cross-Reactive T-Cell Epitopes of SARS-CoV-2 with Human Common Cold Coronaviruses, SARS-CoV, MERS-CoV and Live Attenuated Vaccines Presented by HLA Alleles of Indonesian Population.

Reports on T-cell cross-reactivity against SARS-CoV-2 epitopes in unexposed individuals have been linked with prior exposure to the human common cold coronaviruses (HCCCs). Several studies suggested that cross-reactive T-cells response to live attenuated vaccines (LAVs) such as BCG (Bacillus Calmette-Guérin), OPV (Oral Polio Vaccine), and MMR (measles, mumps, and rubella) can limit the development and severity of COVID-19. This study aims to identify potential cross-reactivity between SARS-CoV-2, HCCCs, and LAVs in the context of T-cell epitopes peptides presented by HLA (Human Leukocyte Antigen) alleles of the Indonesian population. SARS-CoV-2 derived T-cell epitopes were predicted using immunoinformatics tools and assessed for their conservancy, variability, and population coverage. Two fully conserved epitopes with 100% similarity and nine heterologous epitopes with identical T-cell receptor (TCR) contact residues were identified from the ORF1ab fragment of SARS-CoV-2 and all HCCCs. Cross-reactive epitopes from various proteins of SARS-CoV-2 and LAVs were also identified (15 epitopes from BCG, 7 epitopes from MMR, but none from OPV). A majority of the identified epitopes were observed to belong to ORF1ab, further suggesting the vital role of ORF1ab in the coronaviruses family and suggesting it as a candidate for a potential universal coronavirus vaccine that protects against severe disease by inducing cell mediated immunity.

Mechanosignals in abdominal aortic aneurysms.

Cumulative evidence has shown that mechanical and frictional forces exert distinct effects in the multi-cellular aortic layers and play a significant role in the development of abdominal aortic aneurysms (AAA). These mechanical cues collectively trigger signaling cascades relying on mechanosensory cellular hubs that regulate vascular remodeling programs leading to the exaggerated degradation of the extracellular matrix (ECM), culminating in lethal aortic rupture. In this review, we provide an update and summarize the current understanding of the mechanotransduction networks in different cell types during AAA development. We focus on different mechanosensors and stressors that accumulate in the AAA sac and the mechanotransduction cascades that contribute to inflammation, oxidative stress, remodeling, and ECM degradation. We provide perspectives on manipulating this mechano-machinery as a new direction for future research in AAA.

Prevalence and Distribution of Dengue Virus in Aedes aegypti in Yogyakarta City before Deployment of Wolbachia Infected Aedes aegypti.

Indonesia is one of the countries where dengue infection is prevalent. In this study we measure the prevalence and distribution of dengue virus (DENV) DENV-infected Aedes aegypti in Yogyakarta City, Indonesia, during the wet season when high dengue transmission period occurred, as baseline data before implementation of a Wolbachia-infected Aedes aegypti trial for dengue control. We applied One-Step Multiplex Real Time PCR (RT-PCR) for the type-specific-detection of dengue viruses in field-caught adult Aedes aegypti mosquitoes. In a prospective field study conducted from December 2015 to May 2016, adult female Aedes aegypti were caught from selected areas in Yogyakarta City, and then screened by using RT-PCR. During the survey period, 36 (0.12%) mosquitoes from amongst 29,252 female mosquitoes were positive for a DENV type. In total, 22.20% of dengue-positive mosquitoes were DENV-1, 25% were DENV-2, 17% were DENV-3, but none were positive for DENV-4. This study has provided dengue virus infection prevalence in field-caught Aedes aegypti and its circulating serotype in Yogyakarta City before deployment of Wolbachia-infected Aedes aegypti.