Characterization and applications of novel DNA- and RNA-targeting CRISPR effectors

Abstract

The development of molecular tools from natural discovery has played an integral role in the history of modern biology. Genome editing systems derived from the CRISPR bacterial immune system have enabled substantial increases in progress in applications such as forward and reverse genetics, protein tagging, gene therapy, and lineage tracing, among many others. While the initial DNA endonuclease family used in these contexts, Cas9, has demonstrated great utility, the discovery, characterization, and application of new programmable nucleic acid targeting systems could improve upon existing applications and provide new opportunities for the use of molecular tools. Here, we present several new families of single-effector CRISPR enzymes, including Cas12a and Cas13, and demonstrate their value for the targeting of either DNA or RNA in vivo and in vitro, for applications in genome editing, RNA knockdown, RNA editing, and diagnostics. Chapter 1 provides an introduction to the CRISPR adaptive immune system, current approaches in genome editing, and a background of tools for RNA-targeting. Chapter 2 describes the characterization of a novel single-effector CRISPR system, the type V system, and its associated nuclease, Cpf1/Cas12a. We demonstrate the targeting rules for Cpf1 in vitro and in vivo and apply it for genome editing. Chapter 3 details the activity and targeting mechanism of the first RNA targeting CRISPR single-effector protein, C2c2/Cas13a, and reprograms it for targeting phage and transcripts in vivo. It also provides a biochemical characterization of Cas13a and describes the collateral effect, a promiscuous cleavage activity that is activated upon target binding in vitro. Chapter 4 applies the collateral effect of Cas13a to generate a platform for sensitive and specific detection of nucleic acids, SHERLOCK. SHERLOCK is demonstrated for numerous diagnostic applications, including detection of viral and bacterial pathogens, SNP genotyping, and detection of mutations in circulating cell-free DNA. Chapter 5 describes the engineering of the Cas13a system for in vivo RNA targeting in eukaryotic cells, and applies Cas13a as a tool for RNA knockdown, RNA binding, and RNA imaging. Chapter 6 expands the in vivo applications of RNA targeting to the Cas13b and Cas13c families, and applies Cas13b for targeted editing of RNA transcripts in mammalian cells for potential therapeutic approaches. Chapter 7 builds upon the SHERLOCK platform, characterizing the activities of a wide variety of Cas13a and Cas13b orthologs to provide in-sample multiplexing capabilities. It also describes the development of an instrument-free approach for detection based on lateral flow. Chapter 8 provides an outlook on future developments in molecular tool development in DNA- and RNA-targeting systems and suggests new potential sources for molecular tools.