Papers & Patents

2024

Faith DR, Kinnersley M, Brooks DM, Drecktrah D, Hall LS, Luo E, Santiago-Frangos A, Wachter J, Samuels DS, Secor PR.: Characterization and genomic analysis of the Lyme disease spirochete bacteriophage phiBB-1. PLOS Pathogens.

Burman N, Belukhina S, Depardieu F, Wilkinson RA, Skutel M, Santiago-Frangos A, Graham AB, Livenskyi A, Chechenina A, Morozova N, Zahl T, Henriques WS, Buyukyoruk M, Rouillon C, Shyrokova L, Kurata T, Hauryliuk V, Severinov K, Groseille J, Thierry A, Koszul R, Tesson F, Bernheim A, Bikard A*, Wiedenheft B*, Isaev A*.: Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection. bioRxiv.

2023 – Prior to the University of Pennsylvania –

Santiago-Frangos A, Henriques WS, Wiegand T, Gauvin CC, Buyukyoruk M, Graham AB, Wilkinson RA, Triem L, Neselu K, Eng E, Lander GC, Wiedenheft B*.: Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays. Nature Structural & Molecular Biology.

Video I made to try describe the findings in NSMB paper to a general audience!

Wiegand T, Wilkinson R, Santiago-Frangos A, Lynes M, Hatzenpichler R, Wiedenheft B*.: Functional and Phylogenetic Diversity of Cas10 Proteins. The CRISPR Journal.

Cas10 proteins have variable domain compositions, with subtype-specific patterns.

2022

Nemudraia A, Nemudryi A, Buyukyoruk M, Scherffius AM, Zahl TR, Wiegand T, Pandey S, Nichols JE, Hall L, McVey A, Lee HH, Wilkinson RA, Snyder LR, Jones JD, Koutmou KS, Santiago‐Frangos A*, Wiedenheft B*.: Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic. Nature Communications

Type III CRISPR-based RNA concentration enhances sequence-specific detection of RNA.

Patent
Nucleic acid detection using type iii crispr complex.
Blake A. Wiedenheft, Andrew Santiago-Frangos, Anna A. Nemudraia, Artem A. Nemudryi.

Santiago‐Frangos A, Nemudryi A, Nemudraia A, Wiegand T, Nichols JE, Krishna P, Scherffius AM, Zahl TR, Wilkinson RA, Wiedenheft B*.: CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics. Methods.

Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.

Roca J, Santiago‐Frangos A, Woodson SA*.: Diversity of bacterial small RNAs drives competitive strategies for a mutual chaperone. Nature Communications.

Hundreds of bacterial small RNAs (sRNAs) require the Hfq chaperone to regulate mRNA expression. Hfq is limiting, thus competition among sRNAs for binding to Hfq shapes the proteomes of individual cells. To understand how sRNAs compete for a common partner, we present a single-molecule fluorescence platform to simultaneously visualize binding and release of multiple sRNAs with Hfq. We show that RNA residents rarely dissociate on their own. Instead, clashes between residents and challengers on the same face of Hfq cause rapid exchange, whereas RNAs that recognize different surfaces may cohabit Hfq for several minutes before one RNA departs. The prevalence of these pathways depends on the structure of each RNA and how it interacts with Hfq. We propose that sRNA diversity creates many pairwise interactions with Hfq that allow for distinct biological outcomes: active exchange favors fast regulation whereas co-residence of dissimilar RNAs favors target co-recognition or target exclusion.

2021

Santiago‐Frangos A, Buyukyoruk M, Wiegand T, Krishna P, Wiedenheft B*.: Distribution and phasing of sequence motifs that facilitate CRISPR adaptation. Current Biology.

CRISPR-associated proteins (Cas1 and Cas2) integrate foreign DNA at the “leader” end of CRISPR loci. Several CRISPR leader sequences are reported to contain a binding site for a DNA-bending protein called integration host factor (IHF). IHF-induced DNA bending kinks the leader of type I-E CRISPRs, recruiting an upstream sequence motif that helps dock Cas1-2 onto the first repeat of the CRISPR locus. To determine the prevalence of IHF-directed CRISPR adaptation, we analyzed 15,274 bacterial and archaeal CRISPR leaders. These experiments reveal multiple IHF binding sites and diverse upstream sequence motifs in a subset of the I-C, I-E, I-F, and II-C CRISPR leaders. We identify subtype-specific motifs and show that the phase of these motifs is critical for CRISPR adaptation. Collectively, this work clarifies the prevalence and mechanism(s) of IHF-dependent CRISPR adaptation and suggests that leader sequences and adaptation proteins may coevolve under the selective pressures of foreign genetic elements like plasmids or phages.

Santiago-Frangos A, Hall LN, Nemudraia A, Nemudryi A, Krishna P, Wiegand T, Wilkinson RA, Snyder DT, Hedges JF, Cicha C, Lee HH, Graham A, Jutila MA, Taylor MP, Wiedenheft B*.: Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic. Cell Reports Medicine.

Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA to 106 copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min.

Patent
Engineered crispr-cas systems and methods for sensitive and specific diagnostics.
Blake A. Wiedenheft, Andrew Santiago-Frangos, Anna A. Nemudraia, Artem A. Nemudryi.

2020

Hirschi M †, Lu WT †, Santiago-Frangos A, Wilkinson, R, Golden SM, Davidson AR, Lander GC, Wiedenheft B*.: AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex. Nature Communications.

The Csy–AcrIF9 complex binds non-sequence-specific DNA.

Panja S, Małecka EM, Santiago-Frangos A, Woodson SA*.: Quantitative analysis of RNA chaperone activity by native gel electrophoresis and fluorescence spectroscopy. RNA chaperones: Methods and Protocols.

2019

Santiago-Frangos A †, Fröhlich KS †, Jeliazkov JR, Małecka EM, Marino G, Gray JJ, Luisi BF*, Woodson SA*, Hardwick SW*.: Caulobacter Crescentus Hfq Structure Reveals a Conserved Mechanism of RNA Annealing Regulation. Proceedings of the National Academy of Sciences.

Schematic summary of the role of the Cc Hfq CTD. (Upper) Apo Cc Hfq is shown in cyan. For clarity, a single CTD only is shown as purple ribbon for the linker region and as red spheres for the acidic tip residues. Positive core rim residues are shown as dark-blue spheres, spatially occluded by the acidic tip residues. When a suboptimal sRNA substrate is encountered, the positive rim residues remain occluded by the CTD and RNA binding is unfavored. However, when a preferred sRNA substrate is encountered, the CTD is displaced and the sRNA is able to engage with the positive rim residues, ultimately leading to sRNA binding and target gene regulation.

Santiago‐Frangos A, Wiegand T, Wiedenheft B.: Cas9 slide‐and‐seek for phage defense and genome engineering. EMBO Journal.

Cas9 search mechanisms and PAM distributions (A) Schematic representation of Cas9 1-dimensional diffusion (sliding) with probabilities assigned by Globyte et al (2019) (B) Schematic of Cas9 average dwell times (Δτav) reported by Globyte et al (2019) on DNA substrates with different number of PAMs. (C) Physical lengths and PAMs distributions in genomes of Homo sapiens, Streptococcus pyogenes, and bacteriophage A25.

Rollins M.F. †, Chowdhury S. †, Carter J., Golden S.M., Miettinen H.M., Santiago-Frangos A. Faith D, Lawrence CM, Lander GC*, Wiedenheft B*.: Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry. Molecular Cell.

Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the “large” Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 “nuclease recruitment helix” that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

2018

Woodson SA*, Panja S, Santiago-Frangos A. Proteins That Chaperone RNA Regulation. Microbiology Spectrum.

Iterative annealing of RNA by chaperones.

Santiago-Frangos A, & Woodson, SA*. Hfq chaperone brings speed dating to bacterial sRNA. Wiley Interdisciplinary Reviews: RNA.

The chaperone Hfq accelerates match-making between small regulatory RNA and mRNA in bacteria. The Hfq core (cyan) binds U and A rich RNA motifs, recruiting complementary strands to arginine patches (blue). Disordered C-terminal domains (violet) sweep RNA from the core, quickly cycling prospective sRNA–mRNA pairs.

2017

Santiago-Frangos A, Jeliazko JR, Gray JJ, Woodson SA*. Acidic C-terminal domains autoregulate the RNA chaperone Hfq. Elife.

Acidic residues in the Hfq CTD are predicted to bind basic core.

2016

Santiago-Frangos A, Kavita K, Schu DJ, Gottesman S, Woodson SA. C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA. Proceedings of the National Academy of Sciences.

Hfq CTD is required for dsRNA release.

2015

Panja S †, Santiago-Frangos A †, Schu DJ, Gottesman S, Woodson SA. Acidic residues in the Hfq chaperone increase the selectivity of sRNA binding and annealing. Journal of Molecular Biology.

Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.