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Engineering CRISPR-Cas9 Systems to Expand Functionality

  • Author(s): Oakes, Benjamin L.
  • Advisor(s): Savage, David F.
  • Doudna, Jennifer A.
  • et al.
No data is associated with this publication.
Abstract

CRISPR-Cas9 is a RNA-protein complex from adaptive bacterial immune systems that has been repurposed to enable convenient and specific alteration of any genome via the introduction of a double-strand DNA break. By its complementarity to a corresponding DNA sequence, the guide RNA directs the Cas9 protein, a DNA endonuclease, to a target site where it binds to and cleaves the DNA. In this dissertation, the Cas9 ortholog of Streptococcus pyogenes was studied. First, detailed structural insights and knowledge about the biochemical requirements of Cas9-mediated cleavage were exploited to generate a method to efficiently bind a single-stranded RNA target rather than double-stranded DNA. Thus, Cas9 can be used as an RNA-guided RNA-binding protein. Second, methods were devised and implemented to improve and expand the function of Cas9 as an RNA-guided DNA endonuclease, especially means to control its activity spatially and temporally. Systematic unbiased and comprehensive exploration of sites within Cas9 that can tolerate insertions of various protein functionalities, yet retain overall Cas9 activity, had not been undertaken. To engineer more complex and dynamic control over Cas9 action, such as allosteric regulation and location-sensitive molecular scaffolding, I developed a ratiometric analysis protocol that is able to identify one functional Cas9 protein out of tens of millions, which permitted the screening of large libraries of engineered Cas9 proteins. These methods were used to successfully modify Cas9 via the insertion of various protein-protein and protein-ligand binding domains that confer new functionalities. Three distinct types of protein domains were inserted into Cas9 with minimal disruption of its core binding and enzymatic functions. In particular, insertions of the ligand-binding domain of a mammalian estrogen receptor generated a Cas9 derivative whose activity is specifically activated upon addition of the synthetic estrogen 4-hydroxy tamoxifen. This particular Cas9 derivative has no detectable background activity in the absence of the hormone analog, thus providing a fine-tuned mechanism to control genome editing and modification with a small molecule. Overall, the methods described in this thesis can be applied to engineer any programmable DNA-binding protein and thereby confer the ability to be activated or deactivated in response to specific molecular signals.

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This item is under embargo until August 27, 2021.