Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Biochemistry, Biophysics and Molecular Biology



First Advisor

Dipali G. Sashital


CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) genes comprise an RNA-guided adaptive immune system in prokaryotes. Cas proteins can acquire short fragments, called spacers, from the invader DNA or RNA and integrate these spacers into the host genomic CRISPR locus. Once transcribed and processed to short CRISPR RNAs (crRNA), the crRNA spacers can guide Cas surveillance complexes to DNA and/or RNA target sequences, called protospacers, resulting in CRISPR interference through target cleavage. In the type I CRISPR system, an existing protospacer can accelerate new spacer acquisition from the same invader DNA, a process called priming. We have investigated the immune mechanism of the type I-E CRISPR-Cas system in Escherichia coli K12. The research presented in this dissertation has focused on how Cascade searches DNA to locate the target and how crRNA sequence and Cascade conformation control interference and priming activities.

The Cascade surveillance complex must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. We developed a single-molecule fluorescence resonance energy transfer (FRET) assay to directly visualize the Type I-E Cascade surveillance complex searching DNA in real time. We find that Cascade randomly samples DNA through short-lived nonspecific electrostatic contacts and quickly dissociates from dsDNA. Cascade locates its target by first searching for short recognition sequences called protospacer adjacent motifs (PAMs). We find that Cascade dwells longer at PAM sites based on interaction with the PAM recognition motif and a lysine-rich loop in Cse1. In addition, we also identify a motif in the Cas7 backbone subunit that is essential for the searching process. Our findings provide a comprehensive structural and kinetic model for efficient target searching by Cascade.

Once Cascade locates its target, it recruits the trans-acting nuclease Cas3 to trigger CRISPR interference. However, mutations in the PAM or the PAM-proximal region of the protospacer, termed the seed, can block interference and lead to primed adaptation. The importance of the seed region and PAM motif has been studied using a few spacers in Type I-E CRISPR system in E. coli K12. However, it is unknown whether spacer sequence has an effect on the activities of CRISPR system. We have analyzed CRISPR interference and priming using 18 endogenous spacers in E. coli K12 to reexamine the PAM and seed sequence requirements and found that CRISPR interference and priming are strongly influenced by spacer sequence. Our interference data for these 18 spacers also indicate that CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported. We further analyzed spacer sequence-specific tolerance of seed or PAM mutations. Our results indicate that seed and PAM mutational tolerance are highly dependent on spacer sequence. We further show that cytosine residue at the -3 and -2 position of the PAM abolishes both interference and priming, indicating that CRISPR-Cas systems avoid self-spacer targeting by avoiding the final 3 nucleotides of the repeat (CCG). In summary, our findings show that CRISPR activities strongly depend on the spacer sequence and CRISPR-Cas systems avoid self-spacers targeting by avoiding recognize the repeat (CCG). Our studies reveal that some spacer sequences may more readily overcome immune system evasion through invader evolution.

Surprisingly, some PAM mutations have little effect on the equilibrium binding affinity of Cascade, but these mutations still block CRISPR interference. We and other groups have found that Cas3 cannot degrade the Cascade-bound target, suggesting that Cascade may adopt an alternative conformation that blocks Cas3 recruitment when bound to these targets. To test this hypothesis, we developed a novel FRET system to study the conformational dynamics of the Cse1 subunit of Cascade, which recognizes the PAM and recruits Cas3. Our results reveal that Cascade adopts alternative conformations when bound to targets that promote interference or priming in vivo. In addition, we identified Cse1 L1 loop mutations that switch Cascade to the priming conformation, changing the functional outcome of Cascade-target binding from interference to priming even when bound to interference targets. Our results demonstrate that Cascade conformation controls CRISPR immune response following target binding.


Copyright Owner

Chaoyou Xue



File Format


File Size

148 pages