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Principal Investigator: Andrew Santiago-Frangos
Organization: UNIVERSITY OF PENNSYLVANIA
Fiscal Year: 2024
Award: $249,000
Funding agency: National Institute of General Medical Sciences
The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems led to
creative new applications that are transforming science and medicine. However, the rapid discovery of
new CRISPR systems outpaces our understanding of their biological roles in anti-phage defense and their
development for novel applications. To acquire immunity to new phages, CRISPR-associated proteins
(Cas1 and Cas2) integrate fragments of phage DNA ("spacers") at the "leader-end" of the CRISPR locus,
near the transcription start site. But how Cas1-2 recognizes the leader-end of the CRISPR remains poorly
understood. Next, the CRISPR locus is transcribed and processed into "guide RNAs" that are loaded into
surveillance complexes (i.e., Csm complex). Upon sensing viral RNA, the Csm complex makes cyclic
oligonucleotide messengers that regulate CRISPR adaptation and nucleases critical for phage defense.
But the biological roles of many of these immune effectors remain understudied. The 1961 discovery of
regulatory DNA motifs kindled an interest in the "grammatical rules" of DNA motifs that control the storage
and retrieval of genetic information. In Aim 1, I will use a bioinformatic approach to discover DNA motifs in
CRISPR leaders with highly conserved sequences and positions, that I hypothesize regulate key steps in
CRISPR biology such as integration, transcription, or RNA processing. I will determine the role of novel
DNA motifs and host factors in CRISPR integration using in vitro biochemical assays and cryo-EM
structural biology. Nucleotide messengers regulate critical cellular processes across the tree of life,
including anti-viral immune responses, cell morphology, and motility. 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. However,
nucleases represent a fraction of the diversity of enzymatic activities predicted to be activated by
nucleotide messengers. In Aim 2, I will determine a biochemical and structural understanding of novel
immune proteins that I predict to be activated by CRISPR-generated nucleotide messengers.The
long-term objectives of this proposal are to address knowledge gaps in our understanding of how bacterial
CRISPR adaptive immune systems function and are regulated, and to repurpose basic mechanistic
insights for the development of biotech and medical applications.
Terms: <Adaptive Immune System><Address><Archaea><Archaeal Genome><Archaebacteria><Archaeobacteria><Archaeon><Area><Assay><Award><BCAR1><BCAR1 Protein><BCAR1 gene><Bacteria><Bacterial DNA><Bacteriophages><Base Pairing><Bio-Informatics><Bioassay><Biochemical><Biochemistry><Bioinformatics><Biological><Biological Assay><Biological Chemistry><Biology><Biotech><Biotechnology><Breast Cancer Anti-Estrogen Resistance 1 Protein><CKRAS protein><COVID detection><COVID-19 detection><COVID19 detection><CRISPR><CRISPR/Cas system><CRK-Associated Substrate><CRKAS><Cas protein><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Signaling><Cellular Function><Cellular Morphology><Cellular Physiology><Cellular Process><Clustered Regularly Interspaced Short Palindromic Repeats><Complex><Conserved Sequence><Creativeness><Cryo-electron Microscopy><Cryoelectron Microscopy><Cyclicity><DNA><DNA Integration><DNA Sequence><DNA Sequence Analyses><DNA Sequence Analysis><DNA analysis><Deoxyribonucleic Acid><Development><Devices><Diagnostic><Electron Cryomicroscopy><Gene Transcription><Genetic><Genetic Transcription><Genome><Guide RNA><Guilt><Host Factor><Host Factor Protein><Immune><Immune response><Immune signaling><Immune system><Immunes><Immunity><Immunological response><In Vitro><Infection><Integrase><Integration Host Factors><Intracellular Communication and Signaling><Knowledge><Life><Ligands><Mediating><Medical><Medicine><Mission><Motility><NIH><National Institutes of Health><Non-Polyadenylated RNA><Nucleic Acids><Nucleotides><Oligo><Oligonucleotides><Periodicity><Phages><Phase><Position><Positioning Attribute><Process><Proteins><Public Health><RNA><RNA Expression><RNA Gene Products><RNA Processing><Research><Resolution><Retrieval><Rhythmicity><Ribonucleic Acid><Role><SARS-CoV-2 detection><Science><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Specificity><Structure><Subcellular Process><System><Training><Transcription><Transcription Initiation Site><Transcription Process><Transcription Start Site><Trees><United States National Institutes of Health><Viral><acquired immune system><acquired immunity><analyze DNA><anti-microbial><antimicrobial><bacterial virus><biologic><biological signal transduction><cell morphology><coronavirus detection><coronavirus disease 2019 detection><coronavirus disease detection><creativity><cryo-EM><cryoEM><cryogenic electron microscopy><detect COVID><detect COVID-19><detect COVID19><detect SARS-CoV-2><detect coronavirus><detect coronavirus disease><detect severe acute respiratory syndrome coronavirus 2><developmental><experiment><experimental research><experimental study><experiments><gRNA><genetic information><host response><immune system function><immune system response><immunoresponse><innovate><innovation><innovative><insight><interest><invention><microbial><novel><nuclease><oligos><p130 cas protein><p130CAS><resolutions><severe acute respiratory syndrome coronavirus 2 detection><site-specific integration><social role><structural biology><viral RNA><viral detection><virus RNA><virus detection>