Dual-RNA Guided Editing of E. Coli’s EcDnaB Helicase Using the Crispr-Cas9 System

Abstract

Precise manipulation of the genomic DNA is necessary for the characterization and identification of new genes and proteins. New technologies have enabled more facile and precise genomic engineering. Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein method affords precise manipulation of the genome. This system relies on a precise RNA guide (crRNA) that guides the cas9 nuclease to a specific location on the genomic, where it creates a double strand break (DSB). We can take advantage of this adaptive immunological advance and reprogram crRNAs to target whatever gene we like and introduce precise mutations to the genome by offering a designer template to introduce the changes and help the cell remain viable. This method is on the forefront of biochemistry and is being implemented in a numerous eukaryotic systems. However, it has yet to be fully utilized in bacterial genome editing. We are interested in exploring the bacterial helicase EcDnaB. The EcDnaB helicase has been hypothesized to unwind DNA in two fashions; both strands simultaneously or by unwinding one strand while excluding the other. Our lab postulates that there is a steric exclusion of one of the strands as it winds around the exterior of the helicase via electrostatic interactions. We used the CRISPR-Cas9 system to precisely mutate six loci on the EcDnaB gene, corresponding to amino acid residues on the external surface of the helicase, in order to better understand the mechanism of unwinding and to support our proposed method of unwinding. Funding Source: National Science Foundation