Date of Graduation

5-2024

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Christopher E. Nelson

Committee Member

Adam Pare

Second Committee Member

Jeffrey Wolchok

Keywords

CRISPR; Cas9; IPTG

Abstract

Abstract Extensively used for genome editing, CRISPR-Cas9 is a relatively new tool that has made a major impact in the genomic engineering landscape. Due to this, the production of CRISPR proteins that are not commercially available can limit research applications. Here, we present methods for optimizing laboratory scale production of CRISPR proteins including Cas9. The method uses IPTG to induce gene expression of the specified Cas9 and nickel column purification to isolate proteins. CRISPR proteins are validated for purity by SDS-PAGE gel, western blot, and for activity by in vitro digestion. We thoroughly validate and compare the effectiveness of BL21(DE3) bacterial strains of BL21(DE3), BL21(DE3) pLysS, BL21(DE3) StarTM, and BL21(DE3) Rosetta transformed with plasmids encoding SpCas9 and Cystine modified SpCas9 (Cys-Cas9) to test and validate which strain yielded the highest concentration and most functional Cas9 protein. SDS- PAGE Gel and concentration measurements indicated BL21(DE3) pLysS bacteria produced the highest concentration of the SpCas9 protein while BL21(DE3) is the best strain to produce a cysteine modified Cas9. Our comparison shows that BL21(DE3) Rosetta was the least effective strain tested. This work establishes a method for accessing the production of two CRISPR-Cas9 variants with four different bacterial strains.

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