Towards a novel single-cell transcriptome-profiling technique: Investigating the capacity of CRISPR RNAs to prime reverse transcription

Abstract

Single-cell RNA sequencing has revolutionized our understanding of molecular biology and has become a ubiquitous tool in laboratories across the world. However, RNA sequencing library preparation still presents a technological obstacle. To alleviate these challenges, scRNA-seq methods utilize cell-specific transcriptome-profiling methods to allow sequencing readouts of each transcript to retain the molecular memory of the cell from which they are derived. This process enables the pooling of multiple distinct cell samples for parallel library preparation. However, multiplexing methods typically demand that cells are separated into independent indexing environments using time-consuming or resource-intensive methods, thereby presenting a technological challenge. Here we propose a unique barcoding CRISPR-Cas13-based technique that utilizes cellular machinery for transcriptome-profiling, which could dramatically reduce costs and accelerate the library preparation process. This thesis explores the molecular mechanism by which CIRSPR-Cas13 profiles the transcriptome. We show that CRISPR RNAs are sufficient for priming reverse transcription of specific transcripts both in vitro and in situ. We then show the efficiencies of priming reverse transcription by crRNAs across varying reaction conditions. Finally, we demonstrate that CRISPR RNA spacer sequences can be designed to indiscriminately target polyadenylated mRNAs for reverse transcription. In sum, we established the groundwork for continued exploration, optimization, and development of a novel cell-specific transcriptome-profiling mechanism.