Kevin W. Doxzen

Gene therapies are designed to address the root cause of disease. As scientific understanding of disease prevention, diagnosis, and treatment improves in tandem with technological innovation, gene therapies have the potential to become safe and effective treatment options for a wide range of genetic and nongenetic diseases. However, as the medical scope of gene therapies expands, consideration must be given to those who will benefit and what proactive steps must be taken to widen development and access potential, particularly in regions carrying a high disease burden.

(2020). Is This Safe? Addressing Societal Concerns About CRISPR-Edited Foods Without Reinforcing GMO Framing. Environmental Communication. Ahead of Print.

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3′,3′-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

The CRISPR integration complex relies on structural features of DNA to achieve site-selective CRISPR array expansion.

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.

Significance: Argonaute proteins are key effectors of eukaryotic RNA interference and, in prokaryotes, function in host genome defense. We show here that a noncanonical clustered regularly interspaced short palindromic repeats (CRISPR)-associated Marinitoga piezophila Argonaute uses 5′-hydroxylated guide RNAs to recognize and cleave substrates rather than using the 5′-phosphorylated guides used by all other known Argonautes. By identifying other prokaryotic Argonautes with a 5′-hydroxyl RNA preference, our data suggest the evolution of an Argonaute subfamily with unique specificity for a 5′-hydroxylated guide.

Combining microcontact printing with particle image velocimetry, live cell imaging and numerical simulations, we have studied the role of cell density, intercel

Bacterial small RNAs (sRNAs) modulate gene expression by base-pairing with target mRNAs. Many sRNAs require the Sm-like RNA binding protein Hfq as a cofactor. Well-characterized interactions between DsrA sRNA and the rpoS mRNA leader were used to understand how Hfq stimulates sRNA pairing with target mRNAs. DsrA annealing stimulates expression of rpoS by disrupting a secondary structure in the rpoS leader, which otherwise prevents translation. Both RNAs bind Hfq with similar affinity but interact with opposite faces of the Hfq hexamer. Using mutations that block interactions between two of the three components, we demonstrate that Hfq binding to a functionally critical (AAN)₄ motif in rpoS mRNA rescues DsrA binding to a hyperstable rpoS mutant. We also show that Hfq cannot stably bridge the RNAs. Persistent ternary complexes only form when the two RNAs are complementary. Thus, Hfq mainly acts by binding and restructuring the rpoS mRNA. However, Hfq binding to DsrA is needed for maximum annealing in vitro, indicating that transient interactions with both RNAs contribute to the regulatory mechanism.