Jessica R. Ingram

Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by non-homologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing using Cas9 in mammalian cell lines and in mice for all four genes examined up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator like effector nucleases, and to non-mammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.

Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor

Molecular “go” signals reveal their secrets Chemokines are proteins that direct how cells move within the body. For instance, chemokines help immune cells locate invading pathogens and ensure that cells position themselves correctly within a developing organ. Cells detect chemokines through G protein–coupled receptors on their surface; however, the molecular details of how these proteins interact remain unclear (see the Perspective by Standfuss). Qin et al. solved the crystal structure of the chemokine receptor CXCR4 bound to the viral chemokine vMIP-II. Burg et al. solved the crystal structure of a viral chemokine receptor bound to the chemokine domain of CX3CL1. Given the role of chemokines in a number of diseases, these results may help in future drug design. Science, this issue p. 1117, p. 1113; see also p. 1071 The crystal structure of a viral chemokine receptor bound to the chemokine CX3CL1 provides insights into chemokine recognition. [Also see Perspective by Standfuss] Chemokines are small proteins that function as immune modulators through activation of chemokine G protein–coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state–like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

Global identification of peptidase specificity by multiplex substrate profiling

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography–tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Durable antitumor responses to CD47 blockade require adaptive immune stimulation

Significance Therapeutic antitumor antibodies are widely used clinically. CD47 is an antiphagocytic ligand expressed by tumors that binds the inhibitory receptor signal regulatory protein alpha (SIRPα) on phagocytic cells. Interruption of CD47–SIRPα interactions in immunodeficient mice bearing human tumors enhances therapeutic antitumor antibody responses by promoting phagocytosis of antibody-bound tumor cells. Here, we use a novel anti-CD47 single domain antibody, derived from an alpaca, in an immunocompetent mouse model of melanoma and find that, in contrast to immunodeficient models, CD47 blockade alone is insufficient to enhance the effects of antimelanoma antibodies. However, when combined with blockade of programmed death-ligand 1 (PD-L1), an immune receptor that inhibits antitumor T cell responses, we find synergistic activity, suggesting a role for both innate and adaptive inhibitory pathways in the response to therapeutic antibodies. Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47–SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.

How lamina-associated polypeptide 1 (LAP1) activates Torsin

Lamina-associated polypeptide 1 (LAP1) resides at the nuclear envelope and interacts with Torsins, poorly understood endoplasmic reticulum (ER)-localized AAA+ ATPases, through a conserved, perinuclear domain. We determined the crystal structure of the perinuclear domain of human LAP1. LAP1 possesses an atypical AAA+ fold. While LAP1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical AAA+ ATPases is found. Based on modeling and electron microscopic analysis, we propose that LAP1 targets Torsin to the nuclear envelope by forming an alternating, heterohexameric (LAP1-Torsin)3 ring, in which LAP1 acts as the Torsin activator. The experimental data show that mutation of arginine 563 in LAP1 reduces its ability to stimulate TorsinA ATPase hydrolysis. This knowledge may help scientists understand the etiology of DYT1 primary dystonia, a movement disorder caused by a single glutamate deletion in TorsinA. DOI: http://dx.doi.org/10.7554/eLife.03239.001

Crystal structure of a substrate-engaged SecY protein-translocation channel

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

Erratum: Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining (Nature Biotechnology (2015) 33 (538-542))

Nat. Biotechnol. 33, 538–542 (2015); published online 23 March 2015; corrected online 16 April 2015 In the version of this article initially published online, the received date of the paper was given as 17 October 2014; the correct date is 11 July 2014. On p.4, left column, second paragraph, 1 μM Scr7 should have been 1 mM Scr7; in Online Methods, second paragraph, Cas9 mRNA, sgRNA and template oligos were erroneously given in ng/ml rather than ng/μl.

Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.

One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism

Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.

Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells

Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non–small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno–positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.