Proteomic Applications of the CRISPR/Cas13 Technology

Abstract

Proteomics is a major branch in the biological systems study, in addition to other “omics” technologies such as genomics, transcriptomics, metabolomics, etc. High-performance mass spectrometry technologies now provide unprecedented power for investigating the composition, organization, function and regulation of the proteome. Integrating proteomics with other advance biochemical and biophysical methods holds a great promise for elucidating complex biological processes.

CRISPR is a shining star in biology field these years. It's hard to find a revolution pervade faster than CRISPR. CRISPR, originally identified as an adaptive immune system used by microbes to defend themselves against invading viruses, has been reprogrammed for genetic editing in living cells. The recently discovered class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effectors can be engineered for mammalian cell RNA targeting and editing. These include CRISPR/CasRx, a Cas13 family member that could serve as simple and reliable platform for studying RNA biology in mammalian cells and disease treatment. At the same time, we need to elucidate the potential influences of expressing CasRx as an exogenous protein on the recipient cells to address safety concerns. In my thesis work, I integrated the Cas13 technology with proteomics and conducted the following studies：

First, I developed a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leveraged CRISPR/CasRx-based RNA targeting and the proximity labeling strategy to identify binding proteins of specific lncRNAs in the native cellular contexts. This method excludes crosslinking mediated complex co-precipitation or additional molecular engineering which are commonly needed in current methods meanwhile could recover weak and transient protein partners. CARPID was applied to lncRNA XIST and the enriched proteins TAF15 and SNF2L were functionally validated involving in X chromosome inactivation. This method was also performed to MALAT1 and DANCR. Comparison of the CARPID results among the three tested lncRNAs in this study with partially shared subcellular distribution resulted in virtually non-overlapped candidates, demonstrating the high specificity of CARPID method. In addition, I also updated the CARPID method by integrating it with the Orthogonal Organic Phase Separation (OOPS) to capture proteins directly bound to lncRNAs. The method was applied to lncRNA DANCR and the enriched senescence-associated proteins point to its potential novel biological functions.

Second, I conducted a multi-dimensional proteomic study of the effects of CasRx and Cas9 expression on recipient cells. Using state-of-the-art quantitative proteomics, I profiled the interactomes of both Cas proteins, as well as their influence on the proteome of human HEK293T cell line. Integrative analysis of these data revealed distinct responses of recipient cells upon CasRx and Cas9 expression. Notably, the NF-κB pathway, known for its involvement in viral infections and stress responses, was specifically induced by CasRx expression. As a potential mechanism, I observed that CasRx induced formation of abnormal double-strand RNAs in the recipient cells. In addition, analyses of the proteomic response of the recipient cells also pointed to upregulation of N4BP1 that have important functions in NF-κB negative regulation. Taken together, these findings suggest that CasRx expression may induce stress response of the recipient cells.
In conclusion, during my PhD study, I integrated the novel CRISPR/Cas13 technology with proteomics in developing a set of tools that dissect RNA-protein interactions. In addition, I also investigated how cells respond to the exogenous expression of CRISPR/CasRx and Cas9. My findings have potential implications for the proper applications of CRISPR-Cas technologies.

Date of Award	13 Aug 2021
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Liang ZHANG (Supervisor)