Evolutionary pressure to protect against phage-induced lysis and rampant horizontal gene transfer has created a wide repertoire of defensive pathways in bacteria. CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated) systems are adaptive immune pathways that use RNA-guided nucleases to direct cleavage of invading nucleic acids. The programmable nature of these enzymes has enabled a revolution for DNA-targeting applications including gene editing, transcriptional control, and genomic imaging. In addition to DNA-targeting enzymes, specific subtypes of CRISPR-Cas systems recognize and degrade single stranded RNA (ssRNA) substrates. Repurposing these ssRNA-targeting enzymes into biotechnological tools is currently limited by a lack of mechanistic information. In this work, we address this issue by redirecting a well-studied DNA-targeting CRISPR nuclease, Cas9, to ssRNA targets and investigating the enzymatic mechanisms of a novel ssRNA-targeting CRISPR nuclease, Cas13a (formerly C2c2).
Typically, Cas9 ignores ssRNA while searching for dsDNA targets due to ssRNA’s inherent single-stranded structure and lack of a protospacer adjacent motif (PAM). We redirected Cas9 to bind and recognize ssRNA targets through addition of a third component, a target-complementary DNA oligonucleotide or PAMmer, that provides a DNA:RNA hybrid PAM. Using primary microRNAs as a model system, we provide proof-of-concept evidence that Cas9:PAMmer complexes can mediate the isolation and subsequent mass spectrometry analysis of protein complexes bound to specific RNAs.
The complexity of redirecting Cas9 to ssRNA substrates motivated us to investigate CRISPR proteins that natively target RNA. We focused on Cas13a, a predicted ribonuclease from Type VI CRISPR-Cas systems. We discovered that Cas13a possesses two distinct catalytic activities, one for site-specific cleavage of its CRISPR RNA (crRNA) and the second for nonspecific ssRNA degradation activated by target binding. These insights allowed us to establish a revised model for ssRNA-targeting by Type VI CRISPR-Cas systems. Through biochemical characterization of the entire Cas13a protein family, we revealed hidden diversity in substrate preferences and defined orthogonal enzyme subfamilies. These diverse Cas13a homologs can be harnessed in parallel for detection of distinct RNA species within complex mixtures for both bacterial immunity and diagnostic applications. Together, this work presents two novel biotechnological applications of CRISPR-Cas nucleases for RNA isolation and RNA detection.¬¬
|Advisor:||Doudna, Jennifer A.|
|Commitee:||Rio, Donald, Ryan, Kathleen, Schaffer, David|
|School:||University of California, Berkeley|
|Department:||Molecular and Cell Biology|
|School Location:||United States -- California|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology, Biochemistry|
|Keywords:||CRISPR, Cas13a, Cas9, Method development, Rna|
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