Student Theses and Dissertations


Wenyan Jiang

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Marraffini Laboratory


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-associated RNases ensures robust immunity in situations that lead to a slow clearance of the target DNA. Recent work on the type II CRISPR-Cas adaptive immune systems has led to the discovery of Cas9, a dsDNA nuclease whose sequence specificity is programmed by small CRSPR RNAs (crRNAs). In collaboration with Dr. David Bikard, a former colleague, we found when reprogrammed to target the genomes of host bacteria, CRISPR can target and kill the cells. The lethal consequence upon targeting made CRISPR-Cas a novel tool for sequence-specific counter-selection. When combined with editing templates, we demonstrated fast and efficient genome editing in Streptococcus pneumoniae, Escherichia coli (when used in combination with λ-Red recombination) and Staphylococcus aureus. Later, we inactivated the nuclease domains of Cas9, creating a catalytically dead Cas9 (dCas9) which retained DNA binding activity. We demonstrated that dCas9, when programmed with appropriate crRNAs, acted as a transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the elongating RNAP. In addition, a fusion between the ω subunit of the RNA polymerase and dCas9 allowed for programmable transcription activation. The easy programmability and high specificity of crRNA-guided Cas9 and dCas9 greatly facilitates both genome editing and modulation of gene expression, and is likely to substantially advance our capability to decipher gene function in prokaryotes and manipulate them for biotechnological purposes.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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