Boards, Advisory Committees, Professional Organizations

  • Member, The Biophysical Society (2008 - Present)

Professional Education

  • Doctor of Philosophy, Stanford University, Physics (2013)
  • Master of Science, Ecole Centrale Paris, Engineering Physics (2007)
  • Master of Science, Royal Institute of Technology, Engineering Physics (2006)

Lab Affiliations

All Publications

  • High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boyle, E. A., Andreasson, J. O., Chircus, L. M., Sternberg, S. H., Wu, M. J., Guegler, C. K., Doudna, J. A., Greenleaf, W. J. 2017; 114 (21): 5461-5466


    The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.

    View details for DOI 10.1073/pnas.1700557114

    View details for Web of Science ID 000401797800053

    View details for PubMedID 28495970

  • Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA She, R., Chakravarty, A. K., Layton, C. J., Chircus, L. M., Andreasson, J. O., Damaraju, N., McMahon, P. L., Buenrostro, J. D., Jarosz, D. F., Greenleaf, W. J. 2017; 114 (14): 3619-3624


    RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

    View details for DOI 10.1073/pnas.1618370114

    View details for Web of Science ID 000398159000041

    View details for PubMedID 28325876

  • The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load CURRENT BIOLOGY Andreasson, J. O., Shastry, S., Hancock, W. O., Block, S. M. 2015; 25 (9): 1166-1175


    The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length, and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains and not to the neck linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1.

    View details for DOI 10.1016/j.cub.2015.03.013

    View details for Web of Science ID 000353999000023

    View details for PubMedID 25866395

    View details for PubMedCentralID PMC4422762

  • Examining kinesin processivity within a general gating framework ELIFE Andreasson, J. O., Milic, B., Chen, G., Guydosh, N. R., Hancock, W. O., Block, S. M. 2015; 4

    View details for DOI 10.7554/eLife.07403

    View details for Web of Science ID 000356146600001

    View details for PubMedID 25902401

  • Kinesin processivity is gated by phosphate release PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Milic, B., Andreasson, J. O., Hancock, W. O., Block, S. M. 2014; 111 (39): 14136-14140
  • A universal pathway for kinesin stepping NATURE STRUCTURAL & MOLECULAR BIOLOGY Clancy, B. E., Behnke-Parks, W. M., Andreasson, J. O., Rosenfeld, S. S., Block, S. M. 2011; 18 (9): 1020-U79


    Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.

    View details for DOI 10.1038/nsmb.2104

    View details for Web of Science ID 000294551200010

    View details for PubMedID 21841789

  • AN OPTICAL APPARATUS FOR ROTATION AND TRAPPING METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B Gutierrez-Medina, B., Andreasson, J. O., Greenleaf, W. J., Laporta, A., Block, S. M. 2010; 475: 377-404


    We present details of the design, construction, and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles (an "optical torque wrench"). The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator (EOM), which affords improved performance over previous designs. The torque imparted to the trapped particle is assessed by measuring the difference between left- and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop. The limited angular range of the EOM (+/-180 degrees ) is extended by rapidly reversing the polarization once a threshold angle is reached, enabling the torque clamp to function over unlimited, continuous rotations at high bandwidth. In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces (approximately 0.1-100 pN) and torques (approximately 1-10,000 pN nm) in the study of biomolecules. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.

    View details for DOI 10.1016/S0076-6879(10)75015-1

    View details for Web of Science ID 000280733800015

    View details for PubMedID 20627165

  • Precision steering of an optical trap by electro-optic deflection OPTICS LETTERS Valentine, M. T., Guydosh, N. R., Gutierrez-Medina, B., Fehr, A. N., Andreasson, J. O., Block, S. M. 2008; 33 (6): 599-601


    We designed, constructed, and tested a single-beam optical trapping instrument employing twin electro-optic deflectors (EODs) to steer the trap in the specimen plane. Compared with traditional instruments based on acousto-optic deflectors (AODs), EOD-based traps offer a significant improvement in light throughput and a reduction in deflection-angle (pointing) errors. These attributes impart improved force and position resolution, making EOD-based traps a promising alternative for high-precision nanomechanical measurements of biomaterials.

    View details for Web of Science ID 000254907500023

    View details for PubMedID 18347722