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APPLICATION OF CRISPR AS A PLATFORM FOR EMERGING VIRAL INFECTION RESEARCH: PATHOGENESIS AND DIAGNOSTICS
figureposted on 2020-09-04, 04:29 authored by Gi Wai Terance Tsang, Shuofeng YuanShuofeng Yuan, Jasper Fuk Woo ChanJasper Fuk Woo Chan
Genome editing is a powerful tool for forward genetic screens. With the recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, manipulation in human genome has become faster, easier, and more accurate. By adopting and repurposing the CRISPR-Cas9 system through the use of nuclease-deficient Cas9 (dCas9), a wide range of target genes can be upregulated in their endogenous context. In addition to the programmable RNase activity of Cas9, a novel type of enzyme, namely Cas13, exhibits collateral activity after recognition and cleavage of a target transcript. This thesis aimed to exploit these recent advances in CRISPR technology to study the pathogenesis of and develop diagnostic applications for emerging viral infections. In the first part of this thesis, transcriptional activated mammalian cells were challenged with Middle East respiratory syndrome coronavirus (MERS-cov) and then underwent next-generation sequencing to identify candidate antiviral genes.
Through this CRISPR activation screening, I aim to discover antiviral genes that could facilitate cell survival against this highly pathogenic emerging virus.
The second part of this thesis explored the feasibility of using CRISPR technology as a diagnostic assay for nucleic acid detection. When CRISPR effector Cas13a binds with target RNA, it activates nonspecific RNase activity and cleave nearby RNA reporters such as fluorophore. The fluorescent signal could then be quantified to determine the quantity of target sequence in a pool of nucleotides. The combination of Cas13a and recombinase polymerase amplification could serve as a field-deployable, rapid, and sensitive diagnostic tool that helps disease monitoring and genotyping. In this thesis, a novel Japanese encephalitis virus (JEV)-specific crRNA was designed and used in the recently described Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) system for detecting JEV RNA.
In summary, in this thesis, CRISPR activation technology was used to conduct a genome-wide overexpression screen of genes that restrict MERS-CoV infection. Two genes, namely Trim38 and UNC93b1, were identified to exhibit inhibitory activity against MERS-CoV. Using the recombinant Cas13a protein, a novel SHERLOCK assay specifically differentiated JEV RNA from Zika virus RNA was established. These findings highlighted the power of CRISPR technology in the study of viral pathogenesis and diagnostics.