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2. Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants

Author

Arizti-Sanz, Jon, Bradley, A’Doriann, Zhang, Yibin B., Boehm, Chloe K., Freije, Catherine A., Grunberg, Michelle E., Kosoko-Thoroddsen, Tinna-Solveig F., Welch, Nicole L., Pillai, Priya P., Mantena, Sreekar, Kim, Gaeun, Uwanibe, Jessica N., John, Oluwagboadurami G., Eromon, Philomena E., Kocher, Gregory, Gross, Robin, Lee, Justin S., Hensley, Lisa E., MacInnis, Bronwyn L., Johnson, Jeremy, Springer, Michael, Happi, Christian T., Sabeti, Pardis C., and Myhrvold, Cameron

Published
2022
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4. A genome-wide arrayed CRISPR screen identifies PLSCR1 as an intrinsic barrier to SARS-CoV-2 entry that recent virus variants have evolved to resist.

Author

Le Pen, Jérémie, Paniccia, Gabrielle, Kinast, Volker, Moncada-Velez, Marcela, Ashbrook, Alison W., Bauer, Michael, Hoffmann, H.-Heinrich, Pinharanda, Ana, Ricardo-Lax, Inna, Stenzel, Ansgar F., Rosado-Olivieri, Edwin A., Dinnon III, Kenneth H., Doyle, William C., Freije, Catherine A., Hong, Seon-Hui, Lee, Danyel, Lewy, Tyler, Luna, Joseph M., Peace, Avery, and Schmidt, Carltin

Subjects
SARS-CoV-2, COVID-19, GENETIC testing, HUMAN genes, INTERFERONS
Abstract

Interferons (IFNs) play a crucial role in the regulation and evolution of host–virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently. Interferons (IFNs) have a role in the regulation of virus-host interactions. This study uses genome-wide CRISPR knockout screen data to identify 28 genes that impact SARS-CoV-2 infection, including PLSCR1, which restricts spike-mediated SARS-CoV-2 entry independently of its IFN-related role. [ABSTRACT FROM AUTHOR]

Published
2024
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5. A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry

Author

Le Pen, Jeremie, primary, Paniccia, Gabrielle, additional, Bauer, Michael, additional, Hoffmann, H.-Heinrich, additional, Kinast, Volker, additional, Moncada-Velez, Marcela, additional, Pinharanda, Ana, additional, Ricardo-Lax, Inna, additional, Stenzel, Ansgar F, additional, Rosado-Olivieri, Edwin A, additional, Ashbrook, Alison W, additional, Dinnon, Kenneth H, additional, Doyle, William, additional, Freije, Catherine, additional, Hong, Seon-Hui, additional, Lee, Danyel, additional, Lewy, Tyler, additional, Luna, Joseph M, additional, Peace, Avery, additional, Schmidt, Carltin, additional, Schneider, Willia M, additional, Winkler, Roni, additional, Larson, Chloe, additional, McGinn, Timothy, additional, Menezes, Miriam-Rose, additional, Ramos-Espiritu, Lavoisier, additional, Banerjee, Priyam, additional, Poirier, John T, additional, Sanchez-Rivera, Francisco J, additional, Zhang, Qian, additional, Casanova, Jean-Laurent, additional, Carroll, Thomas S, additional, Glickman, J. Fraser, additional, Michailidis, Eleftherios, additional, Razooky, Brandon, additional, MacDonald, Margaret R, additional, and Rice, Charles M, additional

Published
2024
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6. Massively multiplexed nucleic acid detection with Cas13

Author

Ackerman, Cheri M., Myhrvold, Cameron, Thakku, Sri Gowtham, Freije, Catherine A., Metsky, Hayden C., Yang, David K., and Ye, Simon H.

Subjects
Nucleic acids -- Physiological aspects -- Identification and classification, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples.sup.1-3 while simultaneously testing for many pathogens.sup.4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents.sup.7 self-organize in a microwell array.sup.8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health.sup.9-11. CRISPR-based nucleic acid detection is used in a platform that can simultaneously detect 169 human-associated viruses in multiple samples, providing scalable, multiplexed pathogen detection aimed at routine surveillance for public health., Author(s): Cheri M. Ackerman [sup.1] [sup.2] , Cameron Myhrvold [sup.1] [sup.3] , Sri Gowtham Thakku [sup.1] [sup.4] , Catherine A. Freije [sup.1] [sup.5] , Hayden C. Metsky [sup.1] [sup.6] , [...]

Published
2020
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8. Field-deployable viral diagnostics using CRISPR-Cas13

Author

Myhrvold, Cameron, Freije, Catherine A., Gootenberg, Jonathan S., Abudayyeh, Omar O., Metsky, Hayden C., Durbin, Ann F., Kellner, Max J., Tan, Amanda L., Paul, Lauren M., Parham, Leda A., Garcia, Kimberly F., Barnes, Kayla G., Chak, Bridget, Mondini, Adriano, Nogueira, Mauricio L., Isern, Sharon, Michael, Scott F., Lorenzana, Ivette, Yozwiak, Nathan L., MacInnis, Bronwyn L., Bosch, Irene, Gehrke, Lee, Zhang, Feng, and Sabeti, Pardis C.

Published
2018

10. Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection

Author

Zhang, Shen-Ying, Clark, Nathaniel E., Freije, Catherine A., Pauwels, Elodie, Taggart, Allison J., Okada, Satoshi, Mandel, Hanna, Garcia, Paula, Ciancanelli, Michael J., Biran, Anat, Lafaille, Fabien G., Tsumura, Miyuki, Cobat, Aurélie, Luo, Jingchuan, Volpi, Stefano, Zimmer, Bastian, Sakata, Sonoko, Dinis, Alexandra, Ohara, Osamu, Garcia Reino, Eduardo J., Dobbs, Kerry, Hasek, Mary, Holloway, Stephen P., McCammon, Karen, Hussong, Stacy A., DeRosa, Nicholas, Van Skike, Candice E., Katolik, Adam, Lorenzo, Lazaro, Hyodo, Maki, Faria, Emilia, Halwani, Rabih, Fukuhara, Rie, Smith, Gregory A., Galvan, Veronica, Damha, Masad J., Al-Muhsen, Saleh, Itan, Yuval, Boeke, Jef D., Notarangelo, Luigi D., Studer, Lorenz, Kobayashi, Masao, Diogo, Luisa, Fairbrother, William G., Abel, Laurent, Rosenberg, Brad R., Hart, P. John, Etzioni, Amos, and Casanova, Jean-Laurent

Published
2018
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11. Nucleic acid detection with CRISPR-Cas13a/C2c2

Author

Gootenberg, Jonathan S., Abudayyeh, Omar O., Lee, Jeong Wook, Essletzbichler, Patrick, Dy, Aaron J., Joung, Julia, Verdine, Vanessa, Donghia, Nina, Daringer, Nichole M., Freije, Catherine A., Myhrvold, Cameron, Bhattacharyya, Roby P., Livny, Jonathan, Regev, Aviv, Koonin, Eugene V., Hung, Deborah T., Sabeti, Pardis C., Collins, James J., and Zhang, Feng

Published
2017

12. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

Author

Arizti-Sanz, Jon, Freije, Catherine A., Stanton, Alexandra C., Petros, Brittany A., Boehm, Chloe K., Siddiqui, Sameed, Shaw, Bennett M., Adams, Gordon, Kosoko-Thoroddsen, Tinna-Solveig F., Kemball, Molly E., Uwanibe, Jessica N., Ajogbasile, Fehintola V., Eromon, Philomena E., Gross, Robin, Wronka, Loni, Caviness, Katie, Hensley, Lisa E., Bergman, Nicholas H., MacInnis, Bronwyn L., Happi, Christian T., Lemieux, Jacob E., Sabeti, Pardis C., and Myhrvold, Cameron

Published
2020
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13. Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

Author

Barnes, Kayla G., Lachenauer, Anna E., Nitido, Adam, Siddiqui, Sameed, Gross, Robin, Beitzel, Brett, Siddle, Katherine J., Freije, Catherine A., Dighero-Kemp, Bonnie, Mehta, Samar B., Carter, Amber, Uwanibe, Jessica, Ajogbasile, Fehintola, Olumade, Testimony, Odia, Ikponmwosa, Sandi, John Demby, Momoh, Mambu, Metsky, Hayden C., Boehm, Chloe K., Lin, Aaron E., Kemball, Molly, Park, Daniel J., Branco, Luis, Boisen, Matt, Sullivan, Brian, Amare, Mihret F., Tiamiyu, Abdulwasiu B., Parker, Zahra F., Iroezindu, Michael, Grant, Donald S., Modjarrad, Kayvon, Myhrvold, Cameron, Garry, Robert F., Palacios, Gustavo, Hensley, Lisa E., Schaffner, Stephen F., Happi, Christian T., Colubri, Andres, and Sabeti, Pardis C.

Published
2020
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14. An RNA-based system to study hepatitis B virus replication and evaluate antivirals

Author

Yu, Yingpu, primary, Schneider, William M., additional, Kass, Maximilian A., additional, Michailidis, Eleftherios, additional, Acevedo, Ashley, additional, Pamplona Mosimann, Ana L., additional, Bordignon, Juliano, additional, Koenig, Alexander, additional, Livingston, Christine M., additional, van Gijzel, Hardeep, additional, Ni, Yi, additional, Ambrose, Pradeep M., additional, Freije, Catherine A., additional, Zhang, Mengyin, additional, Zou, Chenhui, additional, Kabbani, Mohammad, additional, Quirk, Corrine, additional, Jahan, Cyprien, additional, Wu, Xianfang, additional, Urban, Stephan, additional, You, Shihyun, additional, Shlomai, Amir, additional, de Jong, Ype P., additional, and Rice, Charles M., additional

Published
2023
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16. Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13

Author

Arizti-Sanz, Jon, primary, Bradley, A’Doriann, additional, Zhang, Yibin B., additional, Boehm, Chloe K., additional, Freije, Catherine A., additional, Grunberg, Michelle E., additional, Kosoko-Thoroddsen, Tinna-Solveig F., additional, Welch, Nicole L., additional, Pillai, Priya P., additional, Mantena, Sreekar, additional, Kim, Gaeun, additional, Uwanibe, Jessica N., additional, John, Oluwagboadurami G., additional, Eromon, Philomena E., additional, Kocher, Gregory, additional, Gross, Robin, additional, Lee, Justin S., additional, Hensley, Lisa E., additional, Happi, Christian T., additional, Johnson, Jeremy, additional, Sabeti, Pardis C., additional, and Myhrvold, Cameron, additional

Published
2021
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17. Programmable Inhibition and Detection of RNA Viruses Using Cas13

Author

Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Harvard University--MIT Division of Health Sciences and Technology, Freije, Catherine A, Myhrvold, Cameron, Boehm, Chloe K, Lin, Aaron E, Welch, Nicole L, Carter, Amber, Metsky, Hayden C, Luo, Cynthia Y, Abudayyeh, Omar O, Gootenberg, Jonathan S, Yozwiak, Nathan L, Zhang, Feng, Sabeti, Pardis C, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Harvard University--MIT Division of Health Sciences and Technology, Freije, Catherine A, Myhrvold, Cameron, Boehm, Chloe K, Lin, Aaron E, Welch, Nicole L, Carter, Amber, Metsky, Hayden C, Luo, Cynthia Y, Abudayyeh, Omar O, Gootenberg, Jonathan S, Yozwiak, Nathan L, Zhang, Feng, and Sabeti, Pardis C

Abstract

© 2019 The Authors The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development. Freije et al. demonstrate that Cas13 can be programmed to target and destroy the genomes of diverse mammalian single-stranded RNA viruses. They identify design principles for efficient Cas13 targeting of viral RNA and create companion Cas13-based diagnostics to rapidly measure the effects of Cas13 targeting.

Published
2021

18. Zika virus evolution and spread in the Americas

Author

Metsky, Hayden C., Matranga, Christian B., Wohl, Shirlee, Schaffner, Stephen F., Freije, Catherine A., Winnicki, Sarah M., West, Kendra, Qu, James, Baniecki, Mary Lynn, Gladden-Young, Adrianne, Lin, Aaron E., Tomkins-Tinch, Christopher H., Ye, Simon H., Park, Daniel J., Luo, Cynthia Y., Barnes, Kayla G., Shah, Rickey R., Chak, Bridget, Barbosa-Lima, Giselle, Delatorre, Edson, Vieira, Yasmine R., Paul, Lauren M., Tan, Amanda L., Barcellona, Carolyn M., Porcelli, Mario C., Vasquez, Chalmers, Cannons, Andrew C., Cone, Marshall R., Hogan, Kelly N., Kopp, Edgar W., Anzinger, Joshua J., Garcia, Kimberly F., Parham, Leda A., Ramrez, Rosa M. Glvez, Montoya, Maria C. Miranda, Rojas, Diana P., Brown, Catherine M., Hennigan, Scott, Sabina, Brandon, Scotland, Sarah, Gangavarapu, Karthik, Grubaugh, Nathan D., Oliveira, Glenn, Robles-Sikisaka, Refugio, Rambaut, Andrew, Gehrke, Lee, Smole, Sandra, Halloran, M. Elizabeth, Villar, Luis, Mattar, Salim, Lorenzana, Ivette, Cerbino-Neto, Jose, Valim, Clarissa, Degrave, Wim, Bozza, Patricia T., Gnirke, Andreas, Andersen, Kristian G., Isern, Sharon, Michael, Scott F., Bozza, Fernando A., Souza, Thiago M. L., Bosch, Irene, Yozwiak, Nathan L., MacInnis, Bronwyn L., and Sabeti, Pardis C.

Subjects
America -- Health aspects, Zika virus -- Natural history, Zika virus infection -- Distribution, Company distribution practices, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

Author(s): Hayden C. Metsky [1, 2]; Christian B. Matranga [1]; Shirlee Wohl [1, 3]; Stephen F. Schaffner [1, 3, 4]; Catherine A. Freije [1, 3]; Sarah M. Winnicki [1]; Kendra [...]

Published
2017
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19. Genomic epidemiology reveals multiple introductions of Zika virus into the United States

Author

Grubaugh, Nathan D., Ladner, Jason T., Kraemer, Moritz U. G., Dudas, Gytis, Tan, Amanda L., Gangavarapu, Karthik, Wiley, Michael R., White, Stephen, Thz, Julien, Magnani, Diogo M., Prieto, Karla, Reyes, Daniel, Bingham, Andrea M., Paul, Lauren M., Robles-Sikisaka, Refugio, Oliveira, Glenn, Pronty, Darryl, Barcellona, Carolyn M., Metsky, Hayden C., Baniecki, Mary Lynn, Barnes, Kayla G., Chak, Bridget, Freije, Catherine A., Gladden-Young, Adrianne, Gnirke, Andreas, Luo, Cynthia, MacInnis, Bronwyn, Matranga, Christian B., Park, Daniel J., Qu, James, Schaffner, Stephen F., Tomkins-Tinch, Christopher, West, Kendra L., Winnicki, Sarah M., Wohl, Shirlee, Yozwiak, Nathan L., Quick, Joshua, Fauver, Joseph R., Khan, Kamran, Brent, Shannon E., Reiner, Robert C., Jr, Lichtenberger, Paola N., Ricciardi, Michael J., Bailey, Varian K., Watkins, David I., Cone, Marshall R., Kopp, Edgar W., IV, Hogan, Kelly N., Cannons, Andrew C., Jean, Reynald, Monaghan, Andrew J., Garry, Robert F., Loman, Nicholas J., Faria, Nuno R., Porcelli, Mario C., Vasquez, Chalmers, Nagle, Elyse R., Cummings, Derek A. T., Stanek, Danielle, Rambaut, Andrew, Sanchez-Lockhart, Mariano, Sabeti, Pardis C., Gillis, Leah D., Michael, Scott F., Bedford, Trevor, Pybus, Oliver G., Isern, Sharon, Palacios, Gustavo, and Andersen, Kristian G.

Subjects
Zika virus -- Health aspects -- Genetic aspects, Gene expression -- Health aspects, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

Author(s): Nathan D. Grubaugh [1]; Jason T. Ladner [2]; Moritz U. G. Kraemer [3, 4, 5]; Gytis Dudas [6]; Amanda L. Tan [7]; Karthik Gangavarapu [1]; Michael R. Wiley [2, [...]

Published
2017
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20. XPRIZE SHINE - Paper-based SARS-CoV-2 NP Test v1

Author

Arizti-Sanz, Jon, primary, A. Freije, Catherine, additional, K. Boehm, Chloe, additional, M. Siddiqui, Sameed, additional, M. Goodman, Allen, additional, F. Kosoko-Thoroddsen, Tinna-Solveig, additional, Y. Bradley, A'Doriann, additional, Johnson, Jeremy, additional, C. Sabeti, Pardis, additional, and Myhrvold, Cameron, additional

Published
2020
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22. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2

Author

Arizti-Sanz, Jon, primary, Freije, Catherine A., additional, Stanton, Alexandra C., additional, Boehm, Chloe K., additional, Petros, Brittany A., additional, Siddiqui, Sameed, additional, Shaw, Bennett M., additional, Adams, Gordon, additional, Kosoko-Thoroddsen, Tinna-Solveig F., additional, Kemball, Molly E., additional, Gross, Robin, additional, Wronka, Loni, additional, Caviness, Katie, additional, Hensley, Lisa E., additional, Bergman, Nicholas H., additional, MacInnis, Bronwyn L., additional, Lemieux, Jacob E., additional, Sabeti, Pardis C., additional, and Myhrvold, Cameron, additional

Published
2020
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23. Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

Author

Barnes, Kayla G., primary, Lachenauer, Anna E., additional, Nitido, Adam, additional, Siddiqui, Sameed, additional, Gross, Robin, additional, Beitzel, Brett, additional, Siddle, Katherine J., additional, Freije, Catherine A., additional, Dighero-Kemp, Bonnie, additional, Mehta, Samar, additional, Carter, Amber, additional, Uwanibe, Jessica, additional, Ajogbasile, Fehintola, additional, Olumade, Testimony J., additional, Odia, Ikponmwosa, additional, Sandi, John Demby, additional, Momoh, Mambu, additional, Metsky, Hayden C., additional, Boehm, Chloe K., additional, Lin, Aaron E., additional, Kemball, Molly, additional, Park, Daniel J., additional, Grant, Donald S., additional, Happi, Christian T., additional, Branco, Luis, additional, Boisen, Matt, additional, Sullivan, Brian M., additional, Amare, Mihret, additional, Tiamiyu, Abdulwasiu, additional, Parker, Zahra, additional, Iroezindu, Michael, additional, Modjarrad, Kayvon, additional, Myhrvold, Cameron, additional, Garry, Robert F., additional, Palacios, Gustavo, additional, Hensley, Lisa E., additional, Schaffner, Stephen F., additional, Colubri, Andres, additional, and Sabeti, Pardis C., additional

Published
2020
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25. Field-deployable viral diagnostics using CRISPR-Cas13

Author

McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology. Institute for Medical Engineering & Science, Myhrvold, Cameron, Freije, Catherine A., Gootenberg, Jonathan S, Abudayyeh, Omar O., Metsky, Hayden C., Durbin, Ann F, Kellner, Max J., Tan, Amanda L., Paul, Lauren M., Parham, Leda A., Garcia, Kimberly F., Barnes, Kayla G., Chak, Bridget, Mondini, Adriano, Nogueira, Mauricio L., Isern, Sharon, Michael, Scott F., Lorenzana, Ivette, Yozwiak, Nathan L., MacInnis, Bronwyn L., Bosch, Irene, Gehrke, Lee, Zhang, Feng, Sabeti, Pardis C., McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology. Institute for Medical Engineering & Science, Myhrvold, Cameron, Freije, Catherine A., Gootenberg, Jonathan S, Abudayyeh, Omar O., Metsky, Hayden C., Durbin, Ann F, Kellner, Max J., Tan, Amanda L., Paul, Lauren M., Parham, Leda A., Garcia, Kimberly F., Barnes, Kayla G., Chak, Bridget, Mondini, Adriano, Nogueira, Mauricio L., Isern, Sharon, Michael, Scott F., Lorenzana, Ivette, Yozwiak, Nathan L., MacInnis, Bronwyn L., Bosch, Irene, Gehrke, Lee, Zhang, Feng, and Sabeti, Pardis C.

Abstract

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015–2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms., NIH (Grants AI-100190, 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201)

Published
2020

26. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

Author

Harvard University--MIT Division of Health Sciences and Technology, Arizti-Sanz, Jon, Freije, Catherine A., Stanton, Alexandra C., Petros, Brittany A., Boehm, Chloe K., Siddiqui, Sameed, Shaw, Bennett M., Adams, Gordon, Kosoko-Thoroddsen, Tinna-Solveig F., Kemball, Molly E., Uwanibe, Jessica N., Ajogbasile, Fehintola V., Eromon, Philomena E., Gross, Robin, Wronka, Loni, Caviness, Katie, Hensley, Lisa E., Bergman, Nicholas H., MacInnis, Bronwyn L., Happi, Christian T., Lemieux, Jacob E., Sabeti, Pardis C., Myhrvold, Cameron, Harvard University--MIT Division of Health Sciences and Technology, Arizti-Sanz, Jon, Freije, Catherine A., Stanton, Alexandra C., Petros, Brittany A., Boehm, Chloe K., Siddiqui, Sameed, Shaw, Bennett M., Adams, Gordon, Kosoko-Thoroddsen, Tinna-Solveig F., Kemball, Molly E., Uwanibe, Jessica N., Ajogbasile, Fehintola V., Eromon, Philomena E., Gross, Robin, Wronka, Loni, Caviness, Katie, Hensley, Lisa E., Bergman, Nicholas H., MacInnis, Bronwyn L., Happi, Christian T., Lemieux, Jacob E., Sabeti, Pardis C., and Myhrvold, Cameron

Abstract

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE’s results can be visualized with an in-tube fluorescent readout — reducing contamination risk as amplification reaction tubes remain sealed — and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

Published
2020

27. Massively multiplexed nucleic acid detection using Cas13

Author

Broad Institute of MIT and Harvard, Massachusetts Institute of Technology. Department of Biological Engineering, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Ackerman, Cheri M., Myhrvold, Cameron, Thakku, Sri Gowtham, Freije, Catherine A., Metsky, Hayden C., Yang, David K., Ye, Simon H., Boehm, Chloe K., Kosoko-Thoroddsen, Tinna-Sólveig F., Kehe, Jared, Nguyen, Tien G., Carter, Amber, Kulesa, Anthony, Barnes, John R., Dugan, Vivien G., Hung, Deborah T., Blainey, Paul C., Sabeti, Pardis C., Broad Institute of MIT and Harvard, Massachusetts Institute of Technology. Department of Biological Engineering, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Ackerman, Cheri M., Myhrvold, Cameron, Thakku, Sri Gowtham, Freije, Catherine A., Metsky, Hayden C., Yang, David K., Ye, Simon H., Boehm, Chloe K., Kosoko-Thoroddsen, Tinna-Sólveig F., Kehe, Jared, Nguyen, Tien G., Carter, Amber, Kulesa, Anthony, Barnes, John R., Dugan, Vivien G., Hung, Deborah T., Blainey, Paul C., and Sabeti, Pardis C.

Abstract

The overwhelming majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples while simultaneously testing for many pathogens. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanoliter droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of >4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with ≥10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. CARMEN’s intrinsic multiplexing and throughput capabilities make it practical to scale, as miniaturization decreases reagent cost per test >300-fold. Scalable, highly-multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health. ©2020

Published
2020

28. Metagenomic Sequencing of HIV-1 in the Blood and Female Genital Tract Reveals Little Quasispecies Diversity during Acute Infection

Author

Piantadosi, Anne, Freije, Catherine A, Gosmann, Christina, Ye, Simon, Park, Daniel, Schaffner, Stephen F, Tully, Damien C, Allen, Todd M, Dong, Krista L, Sabeti, Pardis C, and Kwon, Douglas S

Subjects
virus diseases
Abstract

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is associated with a significant bottleneck in the viral quasispecies population, yet the timing of that bottleneck is poorly understood. We characterized HIV-1 diversity in the blood and female genital tract (FGT) within 2 weeks after detection of infection in three women enrolled in a unique prospective cohort in South Africa. We assembled full-length HIV-1 genomes from matched cervicovaginal lavage (CVL) samples and plasma. Deep sequencing allowed us to identify intrahost single-nucleotide variants (iSNVs) and to characterize within-sample HIV-1 diversity. Our results demonstrated very little HIV-1 diversity in the FGT and plasma by the time viremia was detectable. Within each subject, the consensus HIV-1 sequences were identical in plasma and CVL fluid. No iSNV was present at >6% frequency. One subject had 77 low-frequency iSNVs across both CVL fluid and plasma, another subject had 14 iSNVs in only CVL fluid from the earliest time point, and the third subject had no iSNVs in CVL fluid or plasma. Overall, the small amount of diversity that we detected was greater in the FGT than in plasma and declined over the first 2 weeks after viremia was detectable, compatible with a very early HIV-1 transmission bottleneck. To our knowledge, our study represents the earliest genomic analysis of HIV-1 in the FGT after transmission. Further, the use of metagenomic sequencing allowed us to characterize other organisms in the FGT, including commensal bacteria and sexually transmitted infections, highlighting the utility of the method to sequence both HIV-1 and its metagenomic environment.IMPORTANCE Due to error-prone replication, HIV-1 generates a diverse population of viruses within a chronically infected individual. When HIV-1 is transmitted to a new individual, one or a few viruses establish the new infection, leading to a genetic bottleneck in the virus population. Understanding the timing and nature of this bottleneck may provide insight into HIV-1 vaccine design and other preventative strategies. We examined the HIV-1 population in three women enrolled in a unique prospective cohort in South Africa who were followed closely during the earliest stages of HIV-1 infection. We found very little HIV-1 diversity in the blood and female genital tract during the first 2 weeks after virus was detected in the bloodstream. These results are compatible with a very early HIV-1 population bottleneck, suggesting the need to study the HIV-1 population in the female genital tract before virus is detectable in the bloodstream.

Published
2018

29. Metagenomic Sequencing of HIV-1 in the Blood and Female Genital Tract Reveals Little Quasispecies Diversity during Acute Infection

Author

Piantadosi, Anne, primary, Freije, Catherine A., additional, Gosmann, Christina, additional, Ye, Simon, additional, Park, Daniel, additional, Schaffner, Stephen F., additional, Tully, Damien C., additional, Allen, Todd M., additional, Dong, Krista L., additional, Sabeti, Pardis C., additional, and Kwon, Douglas S., additional

Published
2019
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30. Genomic Analysis of Lassa Virus during an Increase in Cases in Nigeria in 2018

Author

Siddle, Katherine J., primary, Eromon, Philomena, additional, Barnes, Kayla G., additional, Mehta, Samar, additional, Oguzie, Judith U., additional, Odia, Ikponmwosa, additional, Schaffner, Stephen F., additional, Winnicki, Sarah M., additional, Shah, Rickey R., additional, Qu, James, additional, Wohl, Shirlee, additional, Brehio, Patrick, additional, Iruolagbe, Christopher, additional, Aiyepada, John, additional, Uyigue, Eghosa, additional, Akhilomen, Patience, additional, Okonofua, Grace, additional, Ye, Simon, additional, Kayode, Tolulope, additional, Ajogbasile, Fehintola, additional, Uwanibe, Jessica, additional, Gaye, Amy, additional, Momoh, Mambu, additional, Chak, Bridget, additional, Kotliar, Dylan, additional, Carter, Amber, additional, Gladden-Young, Adrianne, additional, Freije, Catherine A., additional, Omoregie, Omigie, additional, Osiemi, Blessing, additional, Muoebonam, Ekene B., additional, Airende, Michael, additional, Enigbe, Rachael, additional, Ebo, Benevolence, additional, Nosamiefan, Iguosadolo, additional, Oluniyi, Paul, additional, Nekoui, Mahan, additional, Ogbaini-Emovon, Ephraim, additional, Garry, Robert F., additional, Andersen, Kristian G., additional, Park, Daniel J., additional, Yozwiak, Nathan L., additional, Akpede, George, additional, Ihekweazu, Chikwe, additional, Tomori, Oyewale, additional, Okogbenin, Sylvanus, additional, Folarin, Onikepe A., additional, Okokhere, Peter O., additional, MacInnis, Bronwyn L., additional, Sabeti, Pardis C., additional, and Happi, Christian T., additional

Published
2018
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31. Longitudinal transcriptomic characterization of the immune response to acute hepatitis C virus infection in patients with spontaneous viral clearance

Author

Rosenberg, Brad R., primary, Depla, Marion, additional, Freije, Catherine A., additional, Gaucher, Denis, additional, Mazouz, Sabrina, additional, Boisvert, Maude, additional, Bédard, Nathalie, additional, Bruneau, Julie, additional, Rice, Charles M., additional, and Shoukry, Naglaa H., additional

Published
2018
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32. Genetic Variation at IFNL4 Influences Extrahepatic Interferon-Stimulated Gene Expression in Chronic HCV Patients

Author

Rosenberg, Brad R, primary, Freije, Catherine A, additional, Imanaka, Naoko, additional, Chen, Spencer T, additional, Eitson, Jennifer L, additional, Caron, Rachel, additional, Uhl, Skyler A, additional, Zeremski, Marija, additional, Talal, Andrew, additional, Jacobson, Ira M, additional, Rice, Charles M, additional, and Schoggins, John W, additional

Published
2017
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33. Multiple introductions of Zika virus into the United States revealed through genomic epidemiology

Author

Grubaugh, Nathan D, primary, Ladner, Jason T, additional, Kraemer, Moritz UG, additional, Dudas, Gytis, additional, Tan, Amanda L, additional, Gangavarapu, Karthik, additional, Wiley, Michael R, additional, White, Stephen, additional, Thézé, Julien, additional, Magnani, Diogo M, additional, Prieto, Karla, additional, Reyes, Daniel, additional, Bingham, Andrea, additional, Paul, Lauren M, additional, Robles-Sikisaka, Refugio, additional, Oliveira, Glenn, additional, Pronty, Darryl, additional, Metsky, Hayden C, additional, Baniecki, Mary Lynn, additional, Barnes, Kayla G, additional, Chak, Bridget, additional, Freije, Catherine A, additional, Gladden-Young, Adrianne, additional, Gnirke, Andreas, additional, Luo, Cynthia, additional, MacInnis, Bronwyn, additional, Matranga, Christian B, additional, Park, Daniel J, additional, Qu, James, additional, Schaffner, Stephen F, additional, Tomkins-Tinch, Christopher, additional, West, Kendra L, additional, Winnicki, Sarah M, additional, Wohl, Shirlee, additional, Yozwiak, Nathan L, additional, Quick, Joshua, additional, Fauver, Joseph R, additional, Khan, Kamran, additional, Brent, Shannon E, additional, Reiner, Robert C, additional, Lichtenberger, Paola N, additional, Ricciardi, Michael, additional, Bailey, Varian K, additional, Watkins, David I, additional, Cone, Marshall R, additional, Kopp, Edgar W, additional, Hogan, Kelly N, additional, Cannons, Andrew C, additional, Jean, Reynald, additional, Garry, Robert F, additional, Loman, Nicholas J, additional, Faria, Nuno R, additional, Porcelli, Mario C, additional, Vasquez, Chalmers, additional, Nagle, Elyse R, additional, Cummings, Derek AT, additional, Stanek, Danielle, additional, Rambaut, Andrew, additional, Sanchez-Lockhart, Mariano, additional, Sabeti, Pardis C, additional, Gillis, Leah D, additional, Michael, Scott F, additional, Bedford, Trevor, additional, Pybus, Oliver G, additional, Isern, Sharon, additional, Palacios, Gustavo, additional, and Andersen, Kristian G, additional

Published
2017
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34. Zika virus evolution and spread in the Americas

Author

Institute for Medical Engineering and Science, Broad Institute of MIT and Harvard, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Metsky, Hayden C., Schaffner, Stephen F, Quigley, James Edwin, Ye, Simon Huang, Park, Daniel, Barnes, Kayla, Gehrke, Lee, Valim, Clarissa, Gnirke, Andreas, Andersen, Kristian, Bosch, Irene, Matranga, Christian B., Wohl, Shirlee, Freije, Catherine A., Winnicki, Sarah M., West, Kendra, Baniecki, Mary Lynn, Gladden-Young, Adrianne, Lin, Aaron E., Tomkins-Tinch, Christopher H., Luo, Cynthia Y., Shah, Rickey R., Chak, Bridget, Barbosa-Lima, Giselle, Delatorre, Edson, Vieira, Yasmine R., Paul, Lauren M., Tan, Amanda L., Barcellona, Carolyn M., Porcelli, Mario C., Vasquez, Chalmers, Cannons, Andrew C., Cone, Marshall R., Hogan, Kelly N., Kopp, Edgar W., Anzinger, Joshua J., Garcia, Kimberly F., Parham, Leda A., Ramírez, Rosa M. Gélvez, Montoya, Maria C. Miranda, Rojas, Diana P., Brown, Catherine M., Hennigan, Scott, Sabina, Brandon, Scotland, Sarah, Gangavarapu, Karthik, Grubaugh, Nathan D., Oliveira, Glenn, Robles-Sikisaka, Refugio, Rambaut, Andrew, Smole, Sandra, Halloran, M. Elizabeth, Villar, Luis, Mattar, Salim, Lorenzana, Ivette, Cerbino-Neto, Jose, Degrave, Wim, Bozza, Patricia T., Isern, Sharon, Michael, Scott F., Bozza, Fernando A., Souza, Thiago M. L., Yozwiak, Nathan L., MacInnis, Bronwyn L., Sabeti, Pardis C., Institute for Medical Engineering and Science, Broad Institute of MIT and Harvard, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Metsky, Hayden C., Schaffner, Stephen F, Quigley, James Edwin, Ye, Simon Huang, Park, Daniel, Barnes, Kayla, Gehrke, Lee, Valim, Clarissa, Gnirke, Andreas, Andersen, Kristian, Bosch, Irene, Matranga, Christian B., Wohl, Shirlee, Freije, Catherine A., Winnicki, Sarah M., West, Kendra, Baniecki, Mary Lynn, Gladden-Young, Adrianne, Lin, Aaron E., Tomkins-Tinch, Christopher H., Luo, Cynthia Y., Shah, Rickey R., Chak, Bridget, Barbosa-Lima, Giselle, Delatorre, Edson, Vieira, Yasmine R., Paul, Lauren M., Tan, Amanda L., Barcellona, Carolyn M., Porcelli, Mario C., Vasquez, Chalmers, Cannons, Andrew C., Cone, Marshall R., Hogan, Kelly N., Kopp, Edgar W., Anzinger, Joshua J., Garcia, Kimberly F., Parham, Leda A., Ramírez, Rosa M. Gélvez, Montoya, Maria C. Miranda, Rojas, Diana P., Brown, Catherine M., Hennigan, Scott, Sabina, Brandon, Scotland, Sarah, Gangavarapu, Karthik, Grubaugh, Nathan D., Oliveira, Glenn, Robles-Sikisaka, Refugio, Rambaut, Andrew, Smole, Sandra, Halloran, M. Elizabeth, Villar, Luis, Mattar, Salim, Lorenzana, Ivette, Cerbino-Neto, Jose, Degrave, Wim, Bozza, Patricia T., Isern, Sharon, Michael, Scott F., Bozza, Fernando A., Souza, Thiago M. L., Yozwiak, Nathan L., MacInnis, Bronwyn L., and Sabeti, Pardis C.

Abstract

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention1,2, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests., National Institutes of Health (U.S.) (Grant NIAID U19AI110818)

Published
2017

35. Nucleic acid detection with CRISPR-Cas13a/C2c2

Author

Massachusetts Institute of Technology. Institute for Medical Engineering & Science, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Biology, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research at MIT, Gootenberg, Jonathan S, Abudayyeh, Omar Osama, Dy, Aaron James, Joung, Julia, Daringer, Nichole Marie, Regev, Aviv, Hung, Deborah T, Collins, James J., Zhang, Feng, Lee, Jeong Wook, Essletzbichler, Patrick, Verdine, Vanessa, Donghia, Nina, Freije, Catherine A., Myhrvold, Cameron, Bhattacharyya, Roby P., Livny, Jonathan, Koonin, Eugene V., Pardis, C. Sabeti, Abudayyeh, Omar O., Massachusetts Institute of Technology. Institute for Medical Engineering & Science, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Biology, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research at MIT, Gootenberg, Jonathan S, Abudayyeh, Omar Osama, Dy, Aaron James, Joung, Julia, Daringer, Nichole Marie, Regev, Aviv, Hung, Deborah T, Collins, James J., Zhang, Feng, Lee, Jeong Wook, Essletzbichler, Patrick, Verdine, Vanessa, Donghia, Nina, Freije, Catherine A., Myhrvold, Cameron, Bhattacharyya, Roby P., Livny, Jonathan, Koonin, Eugene V., Pardis, C. Sabeti, and Abudayyeh, Omar O.

Abstract

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications., United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant 1R01-MH110049)

Published
2017

37. Sequence‐informed, Cas13‐based Technologies for RNA Viruses

Author

Freije, Catherine Amanda

Subjects
RNA viruses, CRISPR-Cas13, viral sequencing, diagnostics, viral inhibitors
Abstract

Infectious diseases, many caused by RNA viruses, remain a significant global health threat. Three recent events — the Ebola virus epidemic from 2013–2016, the Zika virus pandemic from 2015–2016, and the ongoing severe acute respiratory syndrome coronavirus 2 pandemic — have illustrated that our ability to diagnose and treat known or newly emerging viral threats is severely limited, largely due to RNA viruses’ high diversity and rapid evolution. However, the highly programmable nature of CRISPR-Cas systems as well as continued discovery of new DNA- and RNA-targeting Cas proteins can accelerate the development of alternative diagnostic and therapeutic approaches. In this dissertation, I demonstrate how growing viral genomic information along with the discovery and use of an RNA-guided, RNA-targeting CRISPR effector protein, Cas13, have enabled the development of technologies that can address unmet diagnostic and therapeutic needs. In Chapter 2, I applied multiplexed amplification methods and next-generation sequencing to generate Zika virus genomic data from patient samples collected during the 2015–2016 pandemic in the Americas. Further, I describe both diagnostic and sequencing challenges associated with low-titer viral infections that this pandemic illuminated. These challenges subsequently motivated me to develop the technologies I describe in the following chapters. In Chapter 3, I developed Cas13-based viral diagnostics that can be used in low-resource settings. These diagnostics are specific, sensitive, user-friendly, and have limited equipment requirements. Because of these advancements, I deployed Cas13-based diagnostics for Zika and dengue viruses in Honduras. In Chapter 4, I harnessed Cas13’s sequence-guided, RNase activity to target and inhibit multiple RNA viruses in mammalian cell culture. I systematically tested hundreds of virus-specific Cas13 CRISPR-RNAs to characterize principles governing Cas13 targeting of viral RNA, and I highlighted the unique capacity of Cas13 to act as an end-to-end system for both detection and destruction of RNA viruses. In addition, I used next-generation sequencing to characterize differences in both the host transcriptome and viral population due to Cas13 targeting. In summary, this dissertation demonstrates the combined power of viral genomics and the RNA-guided, RNA-targeting activity of Cas13 to transform diagnosis and treatment of infectious diseases.

Published
2020

38. An RNA-based system to study hepatitis B virus replication and evaluate antivirals.

Author

Yingpu Yu, Schneider, William M., Kass, Maximilian A., Michailidis, Eleftherios, Acevedo, Ashley, Mosimann, Ana L. Pamplona, Bordignon, Juliano, Koenig, Alexander, Livingston, Christine M., van Gijzel, Hardeep, Yi Ni, Ambrose, Pradeep M., Freije, Catherine A., Mengyin Zhang, Chenhui Zou, Kabbani, Mohammad, Quirk, Corrine, Jahan, Cyprien, Xianfang Wu, and Urban, Stephan

Subjects
*HEPATITIS B virus, *REVERSE transcriptase, *VIRAL replication, *HEPATITIS C
Abstract

The article presents a study which described a method to initiate the replication of hepatitis B virus (HBV), a DNA virus, using synthetic RNA. Topics discussed include a test of whether HBV pregenomic RNA (pgRNA) could initiate the virus life cycle, the ability of HBV launch to identify drug resistance mutations de novo, and synthesis of HBV pgRNA.

Published
2023
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39. Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

Author

Arizti-Sanz J, Bradley A, Zhang YB, Boehm CK, Freije CA, Grunberg ME, Kosoko-Thoroddsen TF, Welch NL, Pillai PP, Mantena S, Kim G, Uwanibe JN, John OG, Eromon PE, Kocher G, Gross R, Lee JS, Hensley LE, Happi CT, Johnson J, Sabeti PC, and Myhrvold C

Abstract

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

Published
2021
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40. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2.

Author

Arizti-Sanz J, Freije CA, Stanton AC, Boehm CK, Petros BA, Siddiqui S, Shaw BM, Adams G, Kosoko-Thoroddsen TF, Kemball ME, Gross R, Wronka L, Caviness K, Hensley LE, Bergman NH, MacInnis BL, Lemieux JE, Sabeti PC, and Myhrvold C

Abstract

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities., Competing Interests: Competing interests: C.A.F., P.C.S., and C.M. are inventors on patent filings related to this work. J.E.L. consults for Sherlock Biosciences, Inc. P.C.S. is a co-founder of, shareholder in, and advisor to Sherlock Biosciences, Inc, as well as a Board member of and shareholder in Danaher Corporation.

Published
2020
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