Viral diseases constitute a major threat to the agricultural production and food security throughout the world. Plants cope with the invading viruses by triggering immune responses and small RNA interference (RNAi) systems. In prokaryotes, CRISPR/Cas systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA viruses. The majority of the plant viruses have RNA genomes. The aim of this study is to test the ability of the newly discovered CRISPR/Cas13a immune system, that targets and cleaves single stranded RNA (ssRNA) in prokaryotes, to provide resistance against RNA viruses in plants. Here, I employ the CRISPR/Cas13a system for molecular interference against Turnip Mosaic Virus (TuMV), a plant RNA virus. The results of this study established the CRISPR/Cas13a as a molecular interference machinery against RNA viruses in plants. Specifically, my data show that the CRISPR/Cas13a machinery is able to interfere with and degrade the TuMV (TuMV-GFP) RNA genome. In conclusion, these data indicate that the CRISPR/Cas13 systems can be employed for engineering interference and durable resistance against RNA viruses in diverse plant species.

<p>  </p> <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>

The development of molecular tools that enable precise manipulation and control of biological systems would allow for a broader understanding of cellular functions and applications in biotechnology, synthetic biology, and therapeutic research. The discovery of CRISPR/Cas systems and the understanding and repurposing of their mechanisms have revolutionized the field of molecular biology. Here, I identified and characterized novel CRISPR/Cas systems and applied them for different in vivo and in vitro applications. In this work, I interrogated various Cas13 effector proteins and identified the most efficient Cas13 effector (CasRx) for in planta applications. I adapted CasRx to engineer plant immunity against different plant RNA viruses. CasRx showed robust activity and specificity against RNA viruses, demonstrating its suitability for studying key questions relating to virus biology. To expand the Cas13 toolbox and enable new applications, I performed a homology search of Cas13 enzymes in prokaryotic genomes and metagenomes, and identified previously uncharacterized, novel CRISPR/Cas13 effector proteins. I first identified and functionally characterized a small size, miniature Cas13 effector (named here as mCas13) and combined it with isothermal amplification to develop a simple and sensitive CRISPR-based SARS-CoV-2 diagnostic platform. In addition, I discovered and biochemically characterized the first known thermostable Cas13 proteins and showed that these thermostable proteins are phylogenetically related. I harnessed the unique features of these thermostable enzymes to develop the first one-pot, RT-LAMP coupled Cas13-based nucleic acid detection assay, which was utilized for highly sensitive, specific, and easily programmable detection of SARS-CoV-2 and other viruses. Lastly, I utilized CRISPR/Cas12a to develop a detection assay of plant ssDNA geminiviruses with easy-to-interpret visual readouts, making it suitable for point-of-use applications. In addition, I leveraged the self vs. non-self-discrimination and pre-crRNA processing capabilities of CRISPR/Cas12a, with the allosteric transcription factors (aTFs)- regulated expression of CRISPR array to engineer a field-deployable small molecule detection platform. I demonstrated the ability of the developed platform to detect different tetracycline antibiotics with high sensitivity and specificity. In conclusion, my work demonstrates that the discovery and characterization of programmable nucleic acid targeting systems could enable their utility for biotechnological innovations, including technologies for inhibition of viral replication and diagnostics.

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.