Methods in molecular biology
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The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. ⋯ Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.
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Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in December 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here we provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. We will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.
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Early detection of cancer and the monitoring of cancer recurrence in treated patients are significant challenges in esophageal squamous cell carcinoma (ESCC). Liquid biopsy is the identification of tumor biomarkers from minimally invasive samples of biological fluids, including urine, blood, stool, saliva, or cerebrospinal fluid. ⋯ These sources of information have the potential to significantly improve the management of patients with ESCC. In this chapter, we detail a method for the isolation of cell-free DNA from blood plasma and DNA associated with exosomes in blood from patients with esophageal squamous cell carcinomas.
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Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. ⋯ Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.
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A critical stage in performing gene editing experiments using the CRISPR/Cas9 system is the design of guide RNA (gRNA). In this chapter, we conduct a review of the current gRNA design rules for maximizing on-target Cas9 activity while minimizing off-target activity. In addition, we present some of the currently available computational tools for gRNA activity prediction and assay design.