Objective Plasma microRNAs are modulated during disease and are emerging biomarkers; they have not been characterized in HIV infection. Using our macaque/simian immunodeficiency virus (SIV) model of HIV, we sought to identify a plasma miRNA profile of acute lentiviral infection, evaluate its relationship with known cellular and viral determinants of lentivirus-associated CNS disease, and explore the potential of miRNAs to predict CNS disease. Design Plasma samples were obtained pre-inoculation and ten days post-inoculation from SIV-infected macaques. Methods Plasma miRNA expression profiles were determined by TaqMan low density array for six individuals. miRNA expression was compared with levels of cytokines, virus, and plasma platelet count. miRNA results were confirmed by single miRNA-specific assays for ten macaques. Nineteen individuals were used to validate a disease prediction test. Results A 45-miRNA signature of acute infection (differential expression with p<0.05 after multiple comparison correction) classified plasma as infected or not. Several differentially expressed miRNAs correlated with CNS disease-associated cytokines IL-6 and CCL2 and included predicted and/or validated regulators of the corresponding mRNAs. miRNAs tracked with viral load and platelet count, also predictors of CNS disease. At least six miRNAs were significantly differentially expressed in individuals with severe versus no CNS disease; in an unweighted expression test, they predicted CNS disease. Conclusions Acute-phase differential expression of plasma miRNAs predicts CNS disease and suggests that CNS damage or predisposition to disease progression begins in the earliest phase of infection. Plasma miRNAs should be investigated further as leading indicators of HIV diseases as early as acute infection.

The GAL4-UAS system is a powerful tool for manipulating gene expression, but its application in C. elegans has not been described. Here we systematically optimize the system’s three main components to develop a temperature-optimized GAL4-UAS system (cGAL) that robustly controls gene expression in C. elegans across 15–25°C. We demonstrate its utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression, and provide a basic driver and effector toolkit.

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.