All Publications

  • Disease-specific tau filaments assemble via polymorphic intermediates. Nature Lovestam, S., Li, D., Wagstaff, J. L., Kotecha, A., Kimanius, D., McLaughlin, S. H., Murzin, A. G., Freund, S. M., Goedert, M., Scheres, S. H. 2024; 625 (7993): 119-125


    Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloidfilament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, beta-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

    View details for DOI 10.1038/s41586-023-06788-w

    View details for PubMedID 38030728

  • Modularity and diversity of target selectors in Tn7 transposons MOLECULAR CELL Faure, G., Saito, M., Benler, S., Peng, I., Wolf, Y. I., Strecker, J., Altae-Tran, H., Neumann, E., Li, D., Makarova, K. S., Macrae, R. K., V. Koonin, E., Zhang, F. 2023; 83 (12): 2122-+


    To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

    View details for DOI 10.1016/j.molcel.2023.05.013

    View details for Web of Science ID 001024573500001

    View details for PubMedID 37267947

    View details for PubMedCentralID PMC10293859

  • RNA-activated protein cleavage with a CRISPR-associated endopeptidase SCIENCE Strecker, J., Demircioglu, F., Li, D., Faure, G., Wilkinson, M. E., Gootenberg, J. S., Abudayyeh, O. O., Nishimasu, H., Macrae, R. K., Zhang, F. 2022; 378 (6622): 874-881


    In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

    View details for DOI 10.1126/science.add7450

    View details for Web of Science ID 000909872400008

    View details for PubMedID 36423276

    View details for PubMedCentralID PMC10028731

  • A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station PLOS ONE Stahl-Rommel, S., Li, D., Sung, M., Li, R., Vijayakumar, A., Atabay, K., Bushkin, G., Castro, C. L., Foley, K. D., Copeland, D., Castro-Wallace, S. L., Saavedra, E., Gleason, E. J., Kraves, S. 2021; 16 (6): e0253403


    As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.

    View details for DOI 10.1371/journal.pone.0253403

    View details for Web of Science ID 000671698800059

    View details for PubMedID 34191829

    View details for PubMedCentralID PMC8244870

  • LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding NATURE BIOTECHNOLOGY Ludwig, K. U., Schmithausen, R. M., Li, D., Jacobs, M. L., Hollstein, R., Blumenstock, K., Liebing, J., Slabicki, M., Ben-Shmuel, A., Israeli, O., Weiss, S., Ebert, T. S., Paran, N., Ruediger, W., Wilbring, G., Feldman, D., Lippke, B., Ishorst, N., Hochfeld, L. M., Beins, E. C., Kaltheuner, I. H., Schmitz, M., Woehler, A., Doehla, M., Sib, E., Jentzsch, M., Borrajo, J. D., Strecker, J., Reinhardt, J., Cleary, B., Geyer, M., Holzel, M., Macrae, R., Noethen, M. M., Hoffmann, P., Exner, M., Regev, A., Zhang, F., Schmid-Burgk, J. L. 2021; 39 (12): 1556-+


    Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

    View details for DOI 10.1038/s41587-021-00966-9

    View details for Web of Science ID 000668064600002

    View details for PubMedID 34188222

    View details for PubMedCentralID PMC8678193

  • Highly Parallel Profiling of Cas9 Variant Specificity MOLECULAR CELL Schmid-Burgk, J. L., Gao, L., Li, D., Gardner, Z., Strecker, J., Lash, B., Zhang, F. 2020; 78 (4): 794-+


    Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

    View details for DOI 10.1016/j.molcel.2020.02.023

    View details for Web of Science ID 000535936200021

    View details for PubMedID 32187529

    View details for PubMedCentralID PMC7370240