On Thursday, September 7th, BGG Graduate Student Adrián Moisés Molina Vargas successfully defended his doctoral thesis: 'Developing Design Strategies for Efficient and Specific CRISPR Cas13 RNA-Targeting Applications'. From Spain, Adrián came to the University of Rochester in the fall of 2018. He pursued his thesis research, studying RNA biology and CRISPR-Cas systems, in the laboratory of his advisor Mitchell R. O'Connell, Ph.D., from the Department of Biochemistry and Biophysics (SMD). As a student, Adrián served in the leadership of the UR's Alliance for Diversity in Science & Engineering. Concluding his doctoral studies on a high note, in the summer of 2023, Adrian’s scientific research was distinguished when he earned a Messersmith fellowship.
Congratulations Dr. Molina Vargas!
Adrián’s thesis abstract can be read below.
ABSTRACT: Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) proteins to target and cleave foreign genetic elements in an RNA-guided manner. The recently described Type VI CRISPR-Cas system contains a single effector ribonuclease, Cas13, that binds and processes a guide-RNA (gRNA), forming an RNA-guided RNA-targeting protein complex. Cas13 has been successfully engineered for potent RNA-knockdown in eukaryotic cells with minimal off-target effects, and other exciting applications described to date include utilizing a nuclease-dead Cas13 (dCas13) as a programmable RNA-binding protein for RNA imaging, RNA- splicing, RNA-detection or RNA-editing applications, among others.
However, the principles of guide-RNA selection for efficient and specific Cas13-gRNA RNA binding remain elusive. Previous work indicates that gRNA choice is important because not all gRNAs yield a robust RNA-targeting, and it is likely that there are different gRNA requirements for RNA-binding compared to RNA-cleavage applications. Furthermore, no predictive models exist for guide-RNA binding efficiency and specificity for Cas13 because no systematic binding screens have been carried out. Given that no transcriptome-wide measurements of Cas13-binding specificity have been attempted so far, it is unknown what the Cas13 off-target binding landscape is for human cells. Addressing this knowledge gap and quantitatively understanding the differences between binding and cleavage specificities is integral to the development of specific Cas13 RNA-binding applications. In this project, we have developed an RNA interactome assay based on eCLIP (enhanced Crosslinking and Immunoprecipitation) that allows to capture Cas13 RNA binding and the crRNA that elicited it, which in turn will help us understand binding specificity of Cas13 in a high-throughput manner.
Moreover, similar gRNA design challenges occur in the CRISPR diagnostics space, as exploration of the principles for gRNA design in the context of Cas13-based diagnostics has been limited, leaving it a case-by-case basis exploration of suitable crRNAs. This is particularly relevant when deploying Cas13 to distinguish a pathogen’s genetic variation or whether a sample is pathological or not. Further progress in the diagnostic field requires a deeper understanding of the biophysical parameters that underlie Cas13 RNA-recognition and activation, which in turn will guide the rational design of more specific Cas13 RNA-diagnostics. By using various methods including mismatch tolerance profiling, gRNA length studies, and structure-guided engineering of Cas13a, we report new strategies and Cas13a variants that yield highly specific discrimination of RNA-targets down to single-nucleotide polymorphisms (SNP). We deployed this novel platform for the detection of single-nucleotide polymorphisms in SARS-CoV-2 variants of concern and showed its potential for disease diagnostics or epidemiological surveillance.
In sum, this work will help complete our understanding of the gRNA requirements for on-target efficiency; but more importantly, it will assist us in uncovering what gRNA features contribute to off-target effects that could hinder the use of Cas13 as a specific RNA-targeting tool. Taken together, my project will bolster efforts to develop a range of RNA-targeting applications that can be readily used as research tools to address knowledge gaps in RNA biology and as potential therapeutics.