PEER REVIEWED PUBLICATIONS AND PREPRINTS
a complete list can be found here
Benjamin A Adler, Marena I Trinidad, Daniel Bellieny-Rabelo, Elaine Zhang, Hannah M Karp, Petr Skopintsev, Brittney Thornton, Rachel F Weissman, Peter H Yoon, Linxing Chen, Tomas Hessler, Amy R Eggers, David A Colognori, Ron Boger, Erin E Doherty, Connor A Tsuchida, Ryan V Tran, Laura Hofman, Honglue Shi, Kevin M Wasko, Zehan Zhou, Chenglong Xia, Muntathar Al-Shimary, Jaymin Patel, Vienna Thomas, Rithu Pattali, Matthew J Kan, Anna Vardapetyan, Alana Yang, Arushi Lahiri, Michaela F Maxwell, Andrew Murdock, Glenn Ramit, Hope R Henderson, Roland W Calvert, Rebecca S Bamert, Audrone Lapinaite, Patrick Pausch, Joshua C Cofsky, Erik J Sontheimer, Blake Wiedenheft, Peter C Fineran, Stan Brouns, Dipali Sashital, Brian C Thomas, Christopher T Brown, Daniela S Goltsman, Rodolphe Barrangou, Virginius Siksnys, Jillian F Banfield, David F Savage, Jennifer A Doudna
Nucleic Acids Research
CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.
Fabienne Benz, Sarah Camara-Wilpert, Jakob Russel, Katharina G. Wandera, Rimvydė Čepaitė, Manuel Ares-Arroyo, José Vicente Gomes-Filho, Frank Englert, Johannes Kuehn, Silvana Gloor, Aline Cuénod, Mònica Aguilà-Sans, Lorrie Maccario, Adrian Egli, Lennart Randau, Patrick Pausch, Eduardo Rocha, Chase L. Beisel, Jonas S. Madsen, David Bikard, Alex R. Hall, Søren J Sørensen, Rafael Pinilla-Redondo
Type IV-A CRISPR-Cas systems are primarily encoded on plasmids and form multi-subunit ribonucleoprotein complexes with unknown biological functions. In contrast to other CRISPR-Cas types, they lack the archetypical CRISPR acquisition module and encode a DinG helicase instead of a nuclease component. Type IV-A3 systems are carried by large conjugative plasmids that often harbor multiple antibiotic-resistance genes. Although their CRISPR array contents suggest a role in inter-plasmid conflicts, this function and the underlying mechanisms have remained unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 CRISPR-Cas system co-opts the type I-E adaptation machinery from its clinical Klebsiella pneumoniae host to update its CRISPR array. Furthermore, we demonstrate that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By targeting plasmid core functions, type IV-A3 can prevent the uptake of incoming plasmids, limit their horizontal transfer, and destabilize co-residing plasmids, altogether supporting type IV-A3’s involvement in plasmid competition. Collectively, our findings shed light on the molecular mechanisms and ecological function of type IV-A3 systems and have broad implications for understanding and countering the spread of antibiotic resistance in clinically relevant strains.
Zheng Li, Zhenhui Zhong, Zhongshou Wu, Patrick Pausch, Basem Al-Shayeb, Jasmine Amerasekera, Jennifer A. Doudna, Steven E. Jacobsen
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.
Basem Al-Shayeb, Petr Skopintsev, Katarzyna M. Soczek, Elizabeth C. Stahl, Zheng Li, Evan Groover, Dylan Smock, Amy R. Eggers, Patrick Pausch, Brady F. Cress, Carolyn J. Huang, Brian Staskawicz, David F. Savage, Steven E. Jacobsen, Jillian F. Banfield, Jennifer A. Doudna
CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.
Joy Y Wang, Patrick Pausch, Jennifer A Doudna.
Nature Reviews Microbiology
CRISPR–Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR–Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR–Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR–Cas systems and mobile genetic elements, including the origins and evolution of CRISPR–Cas systems from DNA transposons, retrotransposons and toxin–antitoxin modules. We discuss how the evolution and structural diversity of CRISPR–Cas systems explain their functional complexity and utility as genome editing tools.
Patrick Pausch, Katarzyna M Soczek, Dominik A Herbst, Connor A Tsuchida, Basem Al-Shayeb, Jillian F Banfield, Eva Nogales, Jennifer A Doudna.
Nature Structural & Molecular Biology
CRISPR–CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
Felix Kaspar, Margarita Seeger, Sarah Westarp, Christoph Köllmann, Anna P Lehmann, Patrick Pausch, Sebastian Kemper, Peter Neubauer, Gert Bange, Anett Schallmey, Daniel Werz, Anke Kurreck.
The growing demand for 4′-modified nucleoside analogs in medicinal and biological chemistry is contrasted by the challenging synthetic access to these molecules and the lack of efficient diversification strategies. Herein, we report the development of a biocatalytic diversification approach based on nucleoside phosphorylases, which allows the straightforward installation of a variety of pyrimidine and purine nucleobases on a 4′-alkylated sugar scaffold. Following the identification of a suitable biocatalyst as well as its characterization with kinetic experiments and docking studies, we systematically explored the equilibrium thermodynamics of this reaction system to enable rational yield prediction in transglycosylation reactions via principles of thermodynamic control. Collectively, this work provides analytical methods and thermodynamic frameworks that outline a general roadmap for the characterization of nucleoside phosphorolysis and transglycosylation systems.
Patrick Pausch, Maha Abdelshahid, Wieland Steinchen, Heinrich Schäfer, Fabio Lino Gratani, Sven-Andreas Freibert, Christiane Wolz, Kürşad Turgay, Daniel N Wilson, Gert Bange.
The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress.
Patrick Pausch, Basem Al-Shayeb, Ezra Bisom-Rapp, Connor A Tsuchida, Zheng Li, Brady F Cress, Gavin J Knott, Steven E Jacobsen, Jillian F Banfield, Jennifer A Doudna.
CRISPR-Cas systems are found widely in prokaryotes, where they provide adaptive immunity against virus infection and plasmid transformation. We describe a minimal functional CRISPR-Cas system, comprising a single ~70-kilodalton protein, CasΦ, and a CRISPR array, encoded exclusively in the genomes of huge bacteriophages. CasΦ uses a single active site for both CRISPR RNA (crRNA) processing and crRNA-guided DNA cutting to target foreign nucleic acids. This hypercompact system is active in vitro and in human and plant cells with expanded target recognition capabilities relative to other CRISPR-Cas proteins. Useful for genome editing and DNA detection but with a molecular weight half that of Cas9 and Cas12a genome-editing enzymes, CasΦ offers advantages for cellular delivery that expand the genome editing toolbox.