CPISPR-CAS: From a Prokaryotic Immune System to a Gene Editing Tool
type: CreativeWork
description: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated genes (cas) encode an adaptive, small-RNA-based immune system that protects prokaryotes from infectious phages and plasmids. CRISPR-Cas systems can be classified into three types based on their cas gene content. My thesis work focused on two parts. First, I investigated the mechanism and function of RNA cleavage in type III CRISPR-Cas immunity. Secondly, I developed a tool to manipulate prokaryotic genomes and gene expression by using an engineered type II CRISPR-Cas system. To date, all three types of CRISPR-Cas systems target DNA. Type III CRISPR-Cas immunity displays an elaborate targeting mechanism and distinguishes itself from type I and type II systems in at least two ways. My previous work in collaboration with other members of the lab helped to discover that the system cleaves both DNA and RNA molecules, and that active transcription across the target is necessary for targeting. Whereas DNA cleavage is required for phage DNA clearance and essential for immunity against infection, the function of RNA cleavage is unknown. It is well established that gene expression of many phages is temporally regulated. Using a type III-A CRISPR-Cas system of Staphylococcus epidermidis as a model, I first identified a new CRISPR-associated RNase, Csm6. The transcriptional requirement for DNA cleavage created a challenge for host bacteria. When the target was located in a late-expressed phage gene, the phage infection cycle can proceed unchecked until the target was transcribed, resulting in a sharp increase of viral genomes in host cells. In this targeting condition, genetic inactivation of the type III-A RNases Csm3 and Csm6 led to the accumulation of the target phage mRNA and abrogated immunity. Csm6 was also required to provide defense in the presence of mutated phage targets, when DNA cleavage efficiency was reduced. My results showed that the degradation of phage transcripts by CRISPR…
abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated genes (cas) encode an adaptive, small-RNA-based immune system that protects prokaryotes from infectious phages and plasmids. CRISPR-Cas systems can be classified into three types based on their cas gene content. My thesis work focused on two parts. First, I investigated the mechanism and function of RNA cleavage in type III CRISPR-Cas immunity. Secondly, I developed a tool to manipulate prokaryotic genomes and gene expression by using an engineered type II CRISPR-Cas system. To date, all three types of CRISPR-Cas systems target DNA. Type III CRISPR-Cas immunity displays an elaborate targeting mechanism and distinguishes itself from type I and type II systems in at least two ways. My previous work in collaboration with other members of the lab helped to discover that the system cleaves both DNA and RNA molecules, and that active transcription across the target is necessary for targeting. Whereas DNA cleavage is required for phage DNA clearance and essential for immunity against infection, the function of RNA cleavage is unknown. It is well established that gene expression of many phages is temporally regulated. Using a type III-A CRISPR-Cas system of Staphylococcus epidermidis as a model, I first identified a new CRISPR-associated RNase, Csm6. The transcriptional requirement for DNA cleavage created a challenge for host bacteria. When the target was located in a late-expressed phage gene, the phage infection cycle can proceed unchecked until the target was transcribed, resulting in a sharp increase of viral genomes in host cells. In this targeting condition, genetic inactivation of the type III-A RNases Csm3 and Csm6 led to the accumulation of the target phage mRNA and abrogated immunity. Csm6 was also required to provide defense in the presence of mutated phage targets, when DNA cleavage efficiency was reduced. My results showed that the degradation of phage transcripts by CRISPR…
author: Jiang, Wenyan
keywords: CRISPR-Cas, RNA cleavage, type III immunity, Csm6, Cas9, prokaryotic genome editing, Life Sciences
date published: 2016-01-01T08:00:00Z
date modified: 2026-07-24T04:11:45Z
provider: Rockefeller
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Rockefeller