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Investigating cellular functions of the SMARCAD1 gene in human MPNST cells by CRISPR-Cas13d knockdownHan Han (12442215) 22 April 2022 (has links)
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<p>Malignant Peripheral Nerve Sheath Tumor (MPNST) is a form of soft tissue sarcoma arising from peripheral nerve sheath cells. Currently, there is no clinically available targeted therapy because the targetable essential driver genes in this tumor are largely unknown. SMARCAD1 (SWI/SNF-related, matrix-associated actin-dependent regulator of chromatin, subfamily A, containing DEAD/H box 1) has been identified as a new tumor suppressor of MPNSTs in zebrafish. Several studies have also linked <em>SMARCAD1</em> with cancer development together. However, the cellular roles of <em>SMARCAD1</em> in human MPNST cells remain unclear. To investigate DNA damage repair functions of SMARCAD1 in human MPNST, we created a doxycycline-inducible Schwannoma cell line by CRISPR-Cas13d, a newly developed mRNA knockdown method. I verified efficiently SMARCAD1 knockdown cell line by western blot. In addition, knockdown of SMARCAD1 inhibits Schwannoma cell proliferation and anchorage-independent growth. It is reported that SMARCAD1 is involved in DNA damage repair mechanisms. I confirmed that loss of SMARCAD1 expression compromises DNA damage repairing function in Schwannoma cells. This result was also verified in two zebrafish <em>smarcad1</em> mutants. In summary, I utilized a novel gene knockdown approach to generate a SMARCAD1 Schwannoma cell line and validated its function in DNA damage repair. This study might provide information for developing a new treatment option for MPNSTs.</p>
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Harnessing a novel compact CRISPR-Cas13b for SARS-CoV-2 diagnosticsWang, Qiaochu 04 1900 (has links)
The outbreak of infectious diseases across the world results in huge disasters for public
health. Rapid and effective diagnostic methods are crucial for disease identification and
transmission control. Since first identified in late 2019, the pandemic of COVID-19
caused by the SARS-CoV-2 virus resulted in unprecedented catastrophe globally. To
control the further spread of COVID-19, there is an urgent need for rapid, accurate,
cost-effective, and efficient diagnostics. Recently, many CRISPR-based diagnostics
have been developed by coupling isothermal amplification methods with Cas proteinmediated
nucleic acid detection. Compared with conventional methods like RT-qPCR,
CRISPR-based assays are more cost-effective and efficient without sacrificing
sensitivity and specificity. Here, I developed a Cas13-based assay for SARS-CoV-2
detection with a novel compact Cas13b protein. In this assay, the Cas13 detection is
combined with RT-LAMP, achieving the detection of viral RNA as low as 4 copies/μl.
By utilizing a simple LED-based visualizer (P51™) instead of a plate reader, the
detection result can be visualized directly without using sophisticated instruments. The
compact Cas13b-mediated viral detection together with P51™-based visualization
enable rapid, sensitive, and portable diagnostics for SARS-CoV-2, showing great
potential in application to point-of-care testing.
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