By Anna Dobson, Yousef Kasim

Faculty Mentor: Dr. April Wynn

Abstract

CRISPR Cas-9 is a genetic engineering technology that is used to edit genomes, such as the bacteria E. coli. The LacY and LacZ genes in E. coli help break down the sugar lactose into glucose and galactose. Genetics students use CRIPSR to edit the wild-type LacZ gene within E.coli to a mutated form that is non-functional and cannot breakdown lactose. For CRISPR to successfully edit the LacZ gene, a plasmid containing the DonorGuide LacZ gene sequences must be transformed into the bacterial cell. BioRad has a commercially available CRISPR kit, from which the genetics lab was adapted. Comparisons between the two protocols were performed to determine efficacy and efficiency of each. The adapted genetics lab protocol produced edited E. coli and while reducing the number of steps required and without the addition of arabinose in the media for growing the E. coli than the commercially available kit.

A second adaptation to this protocol was designed to modify the DonorGuide to be specific for the LacY gene rather than the LacZ gene. This modification would produce a new protocol for creating a mutation in a second gene (LacY) responsible for the breakdown of lactose. DonorGuide plasmids were grown up, purified, and restricted to help isolate the specific sequences around the LacZ gene. By analyzing and sequencing the LacZ DonorGuide plasmid and designing LacY specific primers, we can genetically modify the plasmid to target the LacY gene rather than the LacZ gene.