Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

High levels of lipoprotein(a) in transgenic mice exacerbate atherosclerosis and promote vulnerable plaque features in a sex-specific manner.

BACKGROUND AND AIMS: Despite increased clinical interest in lipoprotein(a) (Lp(a)), many questions remain about the molecular mechanisms by which it contributes to atherosclerotic cardiovascular disease. Existing murine transgenic (Tg) Lp(a) models are limited by low plasma levels of Lp(a) and have not consistently shown a pro-atherosclerotic effect of Lp(a). METHODS: We generated Tg mice expressing both human apolipoprotein(a) (apo(a)) and human apoB-100, with pathogenic levels of plasma Lp(a) (range 87-250 mg/dL). Female and male Lp(a) Tg mice (Tg(LPA+/0;APOB+/0)) and human apoB-100-only controls (Tg(APOB+/0)) (n = 10-13/group) were fed a high-fat, high-cholesterol diet for 12 weeks, with Ldlr knocked down using an antisense oligonucleotide. FPLC was used to characterize plasma lipoprotein profiles. Plaque area and necrotic core size were quantified and immunohistochemical assessment of lesions using a variety of cellular and protein markers was performed. RESULTS: Male and female Tg(LPA+/0;APOB+/0) and Tg(APOB+/0) mice exhibited proatherogenic lipoprotein profiles with increased cholesterol-rich VLDL and LDL-sized particles and no difference in plasma total cholesterol between genotypes. Complex lesions developed in the aortic sinus of all mice. Plaque area (+22%), necrotic core size (+25%), and calcified area (+65%) were all significantly increased in female Tg(LPA+/0;APOB+/0) mice compared to female Tg(APOB+/0) mice. Immunohistochemistry of lesions demonstrated that apo(a) deposited in a similar pattern as apoB-100 in Tg(LPA+/0;APOB+/0) mice. Furthermore, female Tg(LPA+/0;APOB+/0) mice exhibited less organized collagen deposition as well as 42% higher staining for oxidized phospholipids (OxPL) compared to female Tg(APOB+/0) mice. Tg(LPA+/0;APOB+/0) mice had dramatically higher levels of plasma OxPL-apo(a) and OxPL-apoB compared to Tg(APOB+/0) mice, and female Tg(LPA+/0;APOB+/0) mice had higher plasma levels of the proinflammatory cytokine MCP-1 (+3.1-fold) compared to female Tg(APOB+/0) mice. CONCLUSIONS: These data suggest a pro-inflammatory phenotype exhibited by female Tg mice expressing Lp(a) that appears to contribute to the development of more severe lesions with greater vulnerable features.

LPA disruption with AAV-CRISPR potently lowers plasma apo(a) in transgenic mouse model: A proof-of-concept study.

Lipoprotein(a) (Lp(a)) represents a unique subclass of circulating lipoprotein particles and consists of an apolipoprotein(a) (apo(a)) molecule covalently bound to apolipoprotein B-100. The metabolism of Lp(a) particles is distinct from that of low-density lipoprotein (LDL) cholesterol, and currently approved lipid-lowering drugs do not provide substantial reductions in Lp(a), a causal risk factor for cardiovascular disease. Somatic genome editing has the potential to be a one-time therapy for individuals with extremely high Lp(a). We generated an LPA transgenic mouse model expressing apo(a) of physiologically relevant size. Adeno-associated virus (AAV) vector delivery of CRISPR-Cas9 was used to disrupt the LPA transgene in the liver. AAV-CRISPR nearly completely eliminated apo(a) from the circulation within a week. We performed genome-wide off-target assays to determine the specificity of CRISPR-Cas9 editing within the context of the human genome. Interestingly, we identified intrachromosomal rearrangements within the LPA cDNA in the transgenic mice as well as in the LPA gene in HEK293T cells, due to the repetitive sequences within LPA itself and neighboring pseudogenes. This proof-of-concept study establishes the feasibility of using CRISPR-Cas9 to disrupt LPA in vivo, and highlights the importance of examining the diverse consequences of CRISPR cutting within repetitive loci and in the genome globally.

Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA.

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 µL reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity.

Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10 000 guides covering the E. coli chromosome. The use of dominant negative RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.