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  • Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nature methods Hess, G. T., Frésard, L., Han, K., Lee, C. H., Li, A., Cimprich, K. A., Montgomery, S. B., Bassik, M. C. 2016

    Abstract

    Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

    View details for DOI 10.1038/nmeth.4038

    View details for PubMedID 27798611

  • M13 Bacteriophage Display Framework That Allows Sortase-Mediated Modification of Surface-Accessible Phage Proteins BIOCONJUGATE CHEMISTRY Hess, G. T., Cragnolini, J. J., Popp, M. W., Allen, M. A., Dougan, S. K., Spooner, E., Ploegh, H. L., Belcher, A. M., Guimaraes, C. P. 2012; 23 (7): 1478-1487

    Abstract

    We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.

    View details for DOI 10.1021/bc300130z

    View details for Web of Science ID 000306452500015

    View details for PubMedID 22759232

  • Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins. eLife Morgens, D. W., Chan, C., Kane, A. J., Weir, N. R., Li, A., Dubreuil, M. M., Tsui, C. K., Hess, G. T., Lavertu, A., Han, K., Polyakov, N., Zhou, J., Handy, E. L., Alabi, P., Dombroski, A., Yao, D., Altman, R. B., Sello, J. K., Denic, V., Bassik, M. C. 2019; 8

    Abstract

    The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

    View details for DOI 10.7554/eLife.48434

    View details for PubMedID 31674906

  • SLC19A1 Is an Importer of the Immunotransmitter cGAMP. Molecular cell Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C., Li, L. 2019

    Abstract

    2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activateintracellular STING. Using a genome-wide CRISPRscreen, we identified SLC19A1 as the first knownimporter of cGAMP and other CDNs, including theinvestigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

    View details for DOI 10.1016/j.molcel.2019.05.006

    View details for PubMedID 31126740

  • Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nature communications Tycko, J., Wainberg, M., Marinov, G. K., Ursu, O., Hess, G. T., Ego, B. K., Li, A., Truong, A., Trevino, A. E., Spees, K., Yao, D., Kaplow, I. M., Greenside, P. G., Morgens, D. W., Phanstiel, D. H., Snyder, M. P., Bintu, L., Greenleaf, W. J., Kundaje, A., Bassik, M. C. 2019; 10 (1): 4063

    Abstract

    Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

    View details for DOI 10.1038/s41467-019-11955-7

    View details for PubMedID 31492858

  • A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies Nat. Genet. Breslow, D. K., Hoogendoorn, S., Kopp, A. R., Morgens, D. W., Vu, B. K., Han, K., Li, A., Hess, G. T., Bassik, M. C., Chen, J. K., V, N. M. 2018; Epub ahead of print: 460–71

    Abstract

    Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

    View details for DOI 10.1038/s41588-018-0054-7

    View details for PubMedCentralID PMC5862771

  • A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nature genetics Breslow, D. K., Hoogendoorn, S., Kopp, A. R., Morgens, D. W., Vu, B. K., Kennedy, M. C., Han, K., Li, A., Hess, G. T., Bassik, M. C., Chen, J. K., Nachury, M. V. 2018; 50 (3): 460–71

    Abstract

    Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

    View details for PubMedID 29459677

  • Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens NATURE COMMUNICATIONS Morgens, D. W., Wainberg, M., Boyle, E. A., Ursu, O., Araya, C. L., Tsui, C. K., Haney, M. S., Hess, G. T., Han, K., Jeng, E. E., Li, A., Snyder, M. P., Greenleaf, W. J., Kundaje, A., Bassik, M. C. 2017; 8

    Abstract

    CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

    View details for DOI 10.1038/ncomms15178

    View details for PubMedID 28474669

  • Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions NATURE BIOTECHNOLOGY Han, K., Jeng, E. E., Hess, G. T., Morgens, D. W., Li, A., Bassik, M. C. 2017; 35 (5): 463-?

    Abstract

    Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.

    View details for DOI 10.1038/nbt.3834

    View details for Web of Science ID 000400809800021

    View details for PubMedID 28319085

  • Population- and individual- specific regulatory variation in Sardinia NATURE GENETICS Pala, M., Zappala, Z., Marongiu, M., Li, X., Davis, J. R., Cusano, R., Crobu, F., Kukurba, K. R., Gloudemans, M. J., Reinier, F., Berutti, R., Piras, M. G., Mulas, A., Zoledziewska, M., Marongiu, M., Sorokin, E. P., Hess, G. T., Smith, K. S., Busonero, F., Maschio, A., Steri, M., Sidore, C., Sanna, S., Fiorillo, E., Bassik, M. C., Sawcer, S. J., Battle, A., Novembre, J., Jones, C., Angius, A., Abecasis, G. R., Schlessinger, D., Cucca, F., Montgomery, S. B. 2017; 49 (5): 700-?

    Abstract

    Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.

    View details for DOI 10.1038/ng.3840

    View details for Web of Science ID 000400051400010

    View details for PubMedID 28394350

  • The impact of rare variation on gene expression across tissues. Nature Li, X., Kim, Y., Tsang, E. K., Davis, J. R., Damani, F. N., Chiang, C., Hess, G. T., Zappala, Z., Strober, B. J., Scott, A. J., Li, A., Ganna, A., Bassik, M. C., Merker, J. D., Hall, I. M., Battle, A., Montgomery, S. B. 2017; 550 (7675): 239–43

    Abstract

    Rare genetic variants are abundant in humans and are expected to contribute to individual disease risk. While genetic association studies have successfully identified common genetic variants associated with susceptibility, these studies are not practical for identifying rare variants. Efforts to distinguish pathogenic variants from benign rare variants have leveraged the genetic code to identify deleterious protein-coding alleles, but no analogous code exists for non-coding variants. Therefore, ascertaining which rare variants have phenotypic effects remains a major challenge. Rare non-coding variants have been associated with extreme gene expression in studies using single tissues, but their effects across tissues are unknown. Here we identify gene expression outliers, or individuals showing extreme expression levels for a particular gene, across 44 human tissues by using combined analyses of whole genomes and multi-tissue RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project v6p release. We find that 58% of underexpression and 28% of overexpression outliers have nearby conserved rare variants compared to 8% of non-outliers. Additionally, we developed RIVER (RNA-informed variant effect on regulation), a Bayesian statistical model that incorporates expression data to predict a regulatory effect for rare variants with higher accuracy than models using genomic annotations alone. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues and provide an integrative method for interpretation of rare variants in individual genomes.

    View details for PubMedID 29022581

  • Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes. Molecular cell Hess, G. T., Tycko, J., Yao, D., Bassik, M. C. 2017; 68 (1): 26–43

    Abstract

    The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base-editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double-stranded breaks and donor templates) and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base-editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome-editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

    View details for PubMedID 28985508

  • Static and Dynamic DNA Loops form AP-1-Bound Activation Hubs during Macrophage Development. Molecular cell Phanstiel, D. H., Van Bortle, K., Spacek, D., Hess, G. T., Shamim, M. S., Machol, I., Love, M. I., Aiden, E. L., Bassik, M. C., Snyder, M. P. 2017; 67 (6): 1037–48.e6

    Abstract

    The three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops in human monocytes and differentiated macrophages. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer activation of preformed loops together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for activator protein 1 (AP-1)-binding events, suggesting that multi-loop activation hubs involving cell-type-specific transcription factors represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.

    View details for PubMedID 28890333

  • Translation readthrough mitigation NATURE Arribere, J. A., Cenik, E. S., Jain, N., Hess, G. T., Lee, C. H., Bassik, M. C., Fire, A. Z. 2016; 534 (7609): 719-?

    Abstract

    A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic ‘Constant Spring’ haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

    View details for DOI 10.1038/nature18308

    View details for Web of Science ID 000378676000044

    View details for PubMedID 27281202

    View details for PubMedCentralID PMC5054982

  • Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self-Assemble End-to-End Multiphage Structures ACS SYNTHETIC BIOLOGY Hess, G. T., Guimaraes, C. P., Spooner, E., Ploegh, H. L., Belcher, A. M. 2013; 2 (9): 490-496

    Abstract

    M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.

    View details for DOI 10.1021/sb4000195

    View details for Web of Science ID 000324842100002

    View details for PubMedID 23713956

  • Cellular binding, motion, and internalization of synthetic gene delivery polymers BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Hess, G. T., Humphries, W. H., Fay, N. C., Payne, C. K. 2007; 1773 (10): 1583-1588

    Abstract

    Using fluorescence microscopy we have tracked the cellular binding, surface motion, and internalization of polyarginine and polyethylenimine, cationic ligands used for gene and protein delivery. Each ligand was complexed with a quantum dot to provide a photostable probe. Transfection with exogenous DNA was used to relate the observed motion to gene delivery. Cell surface motion was independent of sulfated proteoglycans, but dependent on cholesterol. Cellular internalization required sulfated proteoglycans and cholesterol. These observations suggest that sulfated proteoglycans act as cellular receptors for the cationic ligands, rather than only passive binding sites. Understanding the interaction of polyarginine and polyethylenimine with the plasma membrane may assist in designing more efficient gene delivery systems.

    View details for DOI 10.1016/j.bbamcr.2007.07.009

    View details for Web of Science ID 000250528400009

    View details for PubMedID 17888530

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