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.
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.
Microsatellite instability (MSI) refers to genetic instability in short nucleotide repeats (e.g., 1-6bp tandem repeats), where a cell comprises a different number of repeats as compared to what was inherited from a progenitor cell. This genetic instability is often observed in tumor cells with impaired mismatch repair (MMR), especially in colorectal cancer (CRC) and endometrial cancer. MSI-high tumors can help identify patients that would benefit from additional genetic testing to diagnose Lynch Syndrome. Additionally, MSI can potentially be used as a biomarker to predict response to treatment with immunotherapy and CRC patients with MSI have a significantly better prognosis compared to those with intact mismatch repair. Here we present the VarTrace® MSI Research qPCR Assay, which enables high sensitivity detection of microsatellite instability. The assay uses NuProbe’s PCR-based Blocker Displacement Amplification (BDA) technology to enable the selective amplification of MSI unstable alleles with 1% analytical limit of detection. The assay is intended for the qualitative detection of mutations in 5 MSI loci (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) without the need of matched normal, and reports MSI-high (MSI-H), MSI-low (MSI-L) and Microsatellite Stable (MSS). Analytical validation of the assay has been completed on Horizon MSI/MSS formalin-fixed, paraffin-embedded (FFPE) DNA reference standard, SeraCare MSI AF5% reference panel mix, and 50 CRC FFPE samples. One FFPE section with as little as 2ng of input DNA is enough for this assay. The high sensitivity potentially allows for the detection of MSI status in liquid biopsy. Citation Format: Yan Helen Yan, Blake Young, Zhiheng Wang, Adam Yaseen, Alessandro Pinto, David Zhang. High sensitivity qPCR microsatellite instability detection in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2806.
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