Description of Research Expertise

Research Interests
Determine mechanisms and develop applications of: i) how bacterial and archaeal CRISPR adaptive immune systems make DNA-based memories of past phage infections, and ii) how CRISPR-generated nucleotide messengers signal an immune response.

Keywords
Bacterial & Archaeal Immune Systems, Eukaryotic Immune Systems, CRISPR-Cas, CBASS, Apoptosis, Signal Cascades, Diagnostics, RNA/DNA Integration, Transposition, Reverse Transcription, Cryo-EM, Bioinformatics, Biochemistry, Cryo-EM.

Research Details
Mechanisms and applications of diverse CRISPR integration complexes
Vertebrates, bacteria, and archaea have domesticated transposases (e.g., RAG1 and Cas1) for adaptive immunity. Transposases, integrases and recombinases often co-opt additional DNA-bending proteins (e.g., IHF, HU, H-NS, or HMGB1) that facilitate DNA integration and excision. However, the structural role of DNA folding during this mobilization of DNA remains largely enigmatic. We recently determined a 560 kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1-2/3) and non-Cas proteins (IHF) bind conserved DNA sequence motifs upstream of the CRISPR to fold 150 base-pairs of the genome for site-specific integration of foreign DNA. Going forward we will determine how other bacteria and archaea regulate the integration of RNA/DNA by other CRISPR integrases.

Mechanisms and applications of CRISPR-associated immune signaling pathways
Nucleotides are the most primitive cellular messengers that regulate critical processes across the tree of life, including anti-viral immune responses, cell morphology, and motility. Recent studies show eukaryotic innate immune systems such as cGAS-STING share ancestry with bacterial immune systems. For example, viral RNA-bound Csm and Cmr complexes generate nucleotide messengers (e.g., cA3) that abort infection (e.g., RNA or DNA degradation) by activating immune effector proteins. To address a growing need for rapid and sensitive diagnostics, I co-invented an innovative RNA-guided Csm system for sensitive and sequence-specific detection of SARS-CoV-2 RNA, that repurposes a nuclease immune effector. Computational “guilt-by-association” analyses suggest that CRISPR-generated immune signaling molecules activate a broad range of effectors, and kick-off diverse biochemical cascades – Some of these immune cascades bear a striking similarity to eukaryotic immune pathways. We will determine how diverse signaling cascades mediate immune responses.