Microbial Pathogenesis: Virology

Adam Mefferd
Cullen Lab

Throughout my time as a graduate student in Dr. Cullen’s lab my research has focused on two primary areas. First, I am investigating the use of the DNA editing CRISPR-Cas9 system as a means to target HIV-1. We have shown that by targeting highly conserved regions of HIV-1 (namely the TAR/Tat signaling system) we can effectively inhibit HIV-1 infection by expressing Cas9 and its cognate sgRNA in cells normally permissive for HIV-1 infection. Similarly, we are able to inactivate an integrated HIV-1 provirus in a latently infected cell line by transducing cells with a lentiviral vector expressing Cas9 and well as the cognate sgRNA. Currently, my research on this project focuses on investigating where in the cell Cas9 is cleaving the proviral DNA. Is cleavage occurring before integration or after integration has occurred? We have evidence that in the majority of cases cleavage may be occurring before integration of the provirus takes place. Finally, we are in the process of developing lentiviral vectors to express the CRISPR-Cas9 components in a humanized mouse model of HIV-1.

The second project I have spent much of my time on has been the development of novel technology to facilitate CRISPR-Cas9 vector design. There has been significant innovation in the development of smaller Cas9 proteins that are more amenable to vector packaging limits. However, little progress has been made to design a more compact system for sgRNA expression. Previously, our lab has shown that human tRNAs can be used to express micro RNAs. I have adapted this technology to show that a variety of tRNAs can be used to express sgRNAs at levels comparable to the traditional U6 promoter based expression. The use of tRNAs is advantageous because of their small size (~70 bp) relative to the U6 promoter (~250 bp). This allows us, for example, to include two sgRNA expression cassettes as well as a Cas9 expression cassette within a single AAV vector. Currently, we are adapting this novel technology into both our lentiviral and AAV vectors.