Development of Chimeric Cas9 Nucleases for Accurate and Flexible Genome Editing
Authors
Bolukbasi, Mehmet F.Faculty Advisor
Scot WolfeAcademic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
Molecular, Cell and Cancer BiologyDocument Type
Doctoral DissertationPublication Date
2017-11-30Keywords
CRISPRCas9
specificity
genome editing
precision
programmable DNA-binding domain
enhancer deletion
BCL11a
Biochemistry
Biochemistry, Biophysics, and Structural Biology
Molecular Biology
Metadata
Show full item recordAbstract
There has been tremendous amount of effort focused on the development and improvement of genome editing applications over the decades. Particularly, the development of programmable nucleases has revolutionized genome editing with regards to their improvements in mutagenesis efficacy and targeting feasibility. Programmable nucleases are competent for a variety of genome editing applications. There is growing interest in employing the programmable nucleases in therapeutic genome editing applications, such as correcting mutations in genetic disorders. Type II CRISPR-Cas9 bacterial adaptive immunity systems have recently been engineered as RNA-guided programmable nucleases. Native CRISPR-Cas9 nucleases have two stages of sequence-specific target DNA recognition prior to cleavage: the intrinsic binding of the Cas9 nuclease to a short DNA element (the PAM) followed by testing target site complementarity with the programmable guide RNA. The ease of reprogramming CRISPR-Cas9 nucleases for new target sequences makes them favorable genome editing platform for many applications including gene therapy. However, wild-type Cas9 nucleases have limitations: (i) The PAM element requirement restricts the targeting range of Cas9; (ii) despite the presence of two stages of target recognition, wild-type Cas9 can cleave DNA at unintended sites, which is not desired for therapeutic purposes; and (iii) there is a lack of control over the mutagenic editing product that is procuded. In this study, we developed and characterized chimeric Cas9 platforms to provide solutions to these limitations. In these platforms, the DNA-binding affinity of Cas9 protein from S. pyogenes is attenuated such that the target site binding is dependent on a fused programmable DNA-targeting-unit that recognizes a neighboring DNA-sequence. This modification extends the range of usable PAM elements and substantially improves the targeting specify of wild type Cas9. Furthermore, one of the featured chimeric Cas9 variants developed in this study has both robust nuclease activity and ability to generate predictable uniform editing products. These superior properties of the chimeric Cas9 platforms make them favorable for various genome editing applications and bring programmable nucleases one step closer to therapeutic applications.DOI
10.13028/M26M4QPermanent Link to this Item
http://hdl.handle.net/20.500.14038/32325Rights
Licensed under a Creative Commons licenseDistribution License
http://creativecommons.org/licenses/by-nc/4.0/ae974a485f413a2113503eed53cd6c53
10.13028/M26M4Q
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Except where otherwise noted, this item's license is described as Licensed under a Creative Commons license