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5. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases

Author

Yarnall, Matthew T. N., Ioannidi, Eleonora I., Schmitt-Ulms, Cian, Krajeski, Rohan N., Lim, Justin, Villiger, Lukas, Zhou, Wenyuan, Jiang, Kaiyi, Garushyants, Sofya K., Roberts, Nathaniel, Zhang, Liyang, Vakulskas, Christopher A., Walker, II, John A., Kadina, Anastasia P., Zepeda, Adrianna E., Holden, Kevin, Ma, Hong, Xie, Jun, Gao, Guangping, Foquet, Lander, Bial, Greg, Donnelly, Sara K., Miyata, Yoshinari, Radiloff, Daniel R., Henderson, Jordana M., Ujita, Andrew, Abudayyeh, Omar O., and Gootenberg, Jonathan S.

Published
2023
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6. A global metagenomic map of urban microbiomes and antimicrobial resistance.

Author

Danko, David, Bezdan, Daniela, Afshin, Evan E, Ahsanuddin, Sofia, Bhattacharya, Chandrima, Butler, Daniel J, Chng, Kern Rei, Donnellan, Daisy, Hecht, Jochen, Jackson, Katelyn, Kuchin, Katerina, Karasikov, Mikhail, Lyons, Abigail, Mak, Lauren, Meleshko, Dmitry, Mustafa, Harun, Mutai, Beth, Neches, Russell Y, Ng, Amanda, Nikolayeva, Olga, Nikolayeva, Tatyana, Png, Eileen, Ryon, Krista A, Sanchez, Jorge L, Shaaban, Heba, Sierra, Maria A, Thomas, Dominique, Young, Ben, Abudayyeh, Omar O, Alicea, Josue, Bhattacharyya, Malay, Blekhman, Ran, Castro-Nallar, Eduardo, Cañas, Ana M, Chatziefthimiou, Aspassia D, Crawford, Robert W, De Filippis, Francesca, Deng, Youping, Desnues, Christelle, Dias-Neto, Emmanuel, Dybwad, Marius, Elhaik, Eran, Ercolini, Danilo, Frolova, Alina, Gankin, Dennis, Gootenberg, Jonathan S, Graf, Alexandra B, Green, David C, Hajirasouliha, Iman, Hastings, Jaden JA, Hernandez, Mark, Iraola, Gregorio, Jang, Soojin, Kahles, Andre, Kelly, Frank J, Knights, Kaymisha, Kyrpides, Nikos C, Łabaj, Paweł P, Lee, Patrick KH, Leung, Marcus HY, Ljungdahl, Per O, Mason-Buck, Gabriella, McGrath, Ken, Meydan, Cem, Mongodin, Emmanuel F, Moraes, Milton Ozorio, Nagarajan, Niranjan, Nieto-Caballero, Marina, Noushmehr, Houtan, Oliveira, Manuela, Ossowski, Stephan, Osuolale, Olayinka O, Özcan, Orhan, Paez-Espino, David, Rascovan, Nicolás, Richard, Hugues, Rätsch, Gunnar, Schriml, Lynn M, Semmler, Torsten, Sezerman, Osman U, Shi, Leming, Shi, Tieliu, Siam, Rania, Song, Le Huu, Suzuki, Haruo, Court, Denise Syndercombe, Tighe, Scott W, Tong, Xinzhao, Udekwu, Klas I, Ugalde, Juan A, Valentine, Brandon, Vassilev, Dimitar I, Vayndorf, Elena M, Velavan, Thirumalaisamy P, Wu, Jun, Zambrano, María M, Zhu, Jifeng, Zhu, Sibo, Mason, Christopher E, and International MetaSUB Consortium

Subjects
International MetaSUB Consortium, AMR, BGC, NGS, antimicrobial resistance, built Environment, de novo assembly, global health, metagenome, microbiome, shotgun sequencing, Antimicrobial Resistance, Biotechnology, Human Genome, Genetics, Infection, Developmental Biology, Biological Sciences, Medical and Health Sciences
Abstract

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Published
2021

7. Rapid and accurate species identification for ecological studies and monitoring using CRISPR‐based SHERLOCK

Author

Baerwald, Melinda R, Goodbla, Alisha M, Nagarajan, Raman P, Gootenberg, Jonathan S, Abudayyeh, Omar O, Zhang, Feng, and Schreier, Andrea D

Subjects
Biological Sciences, Ecology, Emerging Infectious Diseases, Animals, Clustered Regularly Interspaced Short Palindromic Repeats, DNA, Fishes, San Francisco, Cas13a, DNA extraction-free, field-based diagnostic, genetic species identification, noninvasive sampling, Osmerid smelt, Evolutionary Biology, Biological sciences
Abstract

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.

Published
2020

8. A transcription factor atlas of directed differentiation

Author

Joung, Julia, Ma, Sai, Tay, Tristan, Geiger-Schuller, Kathryn R., Kirchgatterer, Paul C., Verdine, Vanessa K., Guo, Baolin, Arias-Garcia, Mario A., Allen, William E., Singh, Ankita, Kuksenko, Olena, Abudayyeh, Omar O., Gootenberg, Jonathan S., Fu, Zhanyan, Macrae, Rhiannon K., Buenrostro, Jason D., Regev, Aviv, and Zhang, Feng

Published
2023
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9. Publisher Correction: Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Author

Tycko, Josh, Barrera, Luis A, Huston, Nicholas C, Friedland, Ari E, Wu, Xuebing, Gootenberg, Jonathan S, Abudayyeh, Omar O, Myer, Vic E, Wilson, Christopher J, and Hsu, Patrick D

Subjects
Biomedical and Clinical Sciences, Clinical Sciences, Clinical Research, Good Health and Well Being
Abstract

The original HTML version of this Article incorrectly listed an affiliation of Josh Tycko as 'Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA', instead of the correct 'Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA'. It also incorrectly listed an affiliation of this author as 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA'.The original HTML version incorrectly listed an affiliation of Luis A. Barrera as 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA', instead of the correct 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA 02451, USA'.Finally, the original HTML version incorrectly omitted an affiliation of Nicholas C. Huston: 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA'.This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

Published
2018

10. Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Author

Tycko, Josh, Barrera, Luis A, Huston, Nicholas C, Friedland, Ari E, Wu, Xuebing, Gootenberg, Jonathan S, Abudayyeh, Omar O, Myer, Vic E, Wilson, Christopher J, and Hsu, Patrick D

Subjects
Microbiology, Biological Sciences, Genetics, Human Genome, Biotechnology, Bacterial Proteins, Base Sequence, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Cloning, Molecular, Clustered Regularly Interspaced Short Palindromic Repeats, Gene Editing, Gene Library, Genes, Bacterial, HEK293 Cells, Humans, RNA, Guide, Kinetoplastida, Staphylococcus aureus
Abstract

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

Published
2018

12. Decomposing Oncogenic Transcriptional Signatures to Generate Maps of Divergent Cellular States

Author

Kim, Jong Wook, Abudayyeh, Omar O, Yeerna, Huwate, Yeang, Chen-Hsiang, Stewart, Michelle, Jenkins, Russell W, Kitajima, Shunsuke, Konieczkowski, David J, Medetgul-Ernar, Kate, Cavazos, Taylor, Mah, Clarence, Ting, Stephanie, Van Allen, Eliezer M, Cohen, Ofir, Mcdermott, John, Damato, Emily, Aguirre, Andrew J, Liang, Jonathan, Liberzon, Arthur, Alexe, Gabriella, Doench, John, Ghandi, Mahmoud, Vazquez, Francisca, Weir, Barbara A, Tsherniak, Aviad, Subramanian, Aravind, Meneses-Cime, Karina, Park, Jason, Clemons, Paul, Garraway, Levi A, Thomas, David, Boehm, Jesse S, Barbie, David A, Hahn, William C, Mesirov, Jill P, and Tamayo, Pablo

Subjects
Biological Sciences, Genetics, Biotechnology, Human Genome, Cancer, 2.1 Biological and endogenous factors, Aetiology, Good Health and Well Being, Biomarkers, Tumor, Cell Line, Tumor, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Genes, ras, Genome, Humans, MAP Kinase Signaling System, Neoplasms, Precision Medicine, Bayesian nomogram, cellular states, drug sensitivity, genetic dependency, inferential model, matrix factorization, oncoGPS, oncogenic pathway, precision medicine, transcriptional signatures, Biochemistry and Cell Biology, Biochemistry and cell biology
Abstract

The systematic sequencing of the cancer genome has led to the identification of numerous genetic alterations in cancer. However, a deeper understanding of the functional consequences of these alterations is necessary to guide appropriate therapeutic strategies. Here, we describe Onco-GPS (OncoGenic Positioning System), a data-driven analysis framework to organize individual tumor samples with shared oncogenic alterations onto a reference map defined by their underlying cellular states. We applied the methodology to the RAS pathway and identified nine distinct components that reflect transcriptional activities downstream of RAS and defined several functional states associated with patterns of transcriptional component activation that associates with genomic hallmarks and response to genetic and pharmacological perturbations. These results show that the Onco-GPS is an effective approach to explore the complex landscape of oncogenic cellular states across cancers, and an analytic framework to summarize knowledge, establish relationships, and generate more effective disease models for research or as part of individualized precision medicine paradigms.

Published
2017

14. Programmable RNA writing with trans-splicing

Author

Schmitt-Ulms, Cian, primary, Kayabolen, Alisan, additional, Manero-Carranza, Marcos, additional, Zhou, Nathan, additional, Donnelly, Keira, additional, Nuccio, Sabrina Pia, additional, Kato, Kazuki, additional, Nishimasu, Hiroshi, additional, Gootenberg, Jonathan S., additional, and Abudayyeh, Omar O., additional

Published
2024
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15. Structure and Engineering of Francisella novicida Cas9

Author

Hirano, Hisato, Gootenberg, Jonathan S, Horii, Takuro, Abudayyeh, Omar O, Kimura, Mika, Hsu, Patrick D, Nakane, Takanori, Ishitani, Ryuichiro, Hatada, Izuho, Zhang, Feng, Nishimasu, Hiroshi, and Nureki, Osamu

Subjects
Biochemistry and Cell Biology, Biological Sciences, Genetics, Biotechnology, Animals, Bacterial Proteins, Blastocyst, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Crystallography, X-Ray, Embryo, Mammalian, Endonucleases, Francisella, Genetic Engineering, Mice, Microinjections, Models, Molecular, RNA, Guide, Kinetoplastida, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences
Abstract

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.

Published
2016

16. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA

Author

Patchsung, Maturada, Jantarug, Krittapas, Pattama, Archiraya, Aphicho, Kanokpol, Suraritdechachai, Surased, Meesawat, Piyachat, Sappakhaw, Khomkrit, Leelahakorn, Nattawat, Ruenkam, Theerawat, Wongsatit, Thanakrit, Athipanyasilp, Niracha, Eiamthong, Bhumrapee, Lakkanasirorat, Benya, Phoodokmai, Thitima, Niljianskul, Nootaree, Pakotiprapha, Danaya, Chanarat, Sittinan, Homchan, Aimorn, Tinikul, Ruchanok, Kamutira, Philaiwarong, Phiwkaow, Kochakorn, Soithongcharoen, Sahachat, Kantiwiriyawanitch, Chadaporn, Pongsupasa, Vinutsada, Trisrivirat, Duangthip, Jaroensuk, Juthamas, Wongnate, Thanyaporn, Maenpuen, Somchart, Chaiyen, Pimchai, Kamnerdnakta, Sirichai, Swangsri, Jirawat, Chuthapisith, Suebwong, Sirivatanauksorn, Yongyut, Chaimayo, Chutikarn, Sutthent, Ruengpung, Kantakamalakul, Wannee, Joung, Julia, Ladha, Alim, Jin, Xin, Gootenberg, Jonathan S., Abudayyeh, Omar O., Zhang, Feng, Horthongkham, Navin, and Uttamapinant, Chayasith

Published
2020
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17. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Author

Konermann, Silvana, Brigham, Mark D, Trevino, Alexandro E, Joung, Julia, Abudayyeh, Omar O, Barcena, Clea, Hsu, Patrick D, Habib, Naomi, Gootenberg, Jonathan S, Nishimasu, Hiroshi, Nureki, Osamu, and Zhang, Feng

Subjects
Genetics, Biotechnology, Human Genome, Cancer, Development of treatments and therapeutic interventions, 5.1 Pharmaceuticals, CRISPR-Associated Proteins, CRISPR-Cas Systems, Cell Line, Tumor, Clustered Regularly Interspaced Short Palindromic Repeats, DNA, Complementary, Drug Resistance, Neoplasm, Gene Expression Regulation, Neoplastic, Gene Library, Genetic Engineering, Genetic Loci, Genetic Testing, Genome, Human, Humans, Indoles, Melanoma, Proto-Oncogene Proteins B-raf, RNA, Untranslated, Reproducibility of Results, Sulfonamides, Transcriptional Activation, Up-Regulation, General Science & Technology
Abstract

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

Published
2015

20. Engineered CRISPR-Cas9 nuclease with expanded targeting space

Author

Nishimasu, Hiroshi, Shi, Xi, Ishiguro, Soh, Gao, Linyi, Hirano, Seiichi, Okazaki, Sae, Noda, Taichi, Abudayyeh, Omar O., Gootenberg, Jonathan S., Mori, Hideto, Oura, Seiya, Holmes, Benjamin, Tanaka, Mamoru, Seki, Motoaki, Hirano, Hisato, Aburatani, Hiroyuki, Ishitani, Ryuichiro, Ikawa, Masahito, Yachie, Nozomu, Zhang, Feng, and Nureki, Osamu

Published
2018

22. 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

24. RNA editing with CRISPR-Cas13

Author

Cox, David B. T., Gootenberg, Jonathan S., Abudayyeh, Omar O., Franklin, Brian, Kellner, Max J., Joung, Julia, and Zhang, Feng

Published
2017

25. 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

27. Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses

Author

Jiang, Kaiyi, Lim, Justin, Sgrizzi, Samantha, Trinh, Michael, Kayabolen, Alisan, Yutin, Natalya, Koonin, Eugene V., Abudayyeh, Omar O., and Gootenberg, Jonathan S.

Subjects
Article
Abstract

TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements. Reconstruction of the evolution of these nucleases, which we rename Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems (HERMES), revealed multiple acquisitions of TnpBs by eukaryotes and subsequent diversification. In their adaptation and spread in eukaryotes, HERMES proteins acquired nuclear localization signals, and genes captured introns, indicating extensive, long term adaptation to functioning in eukaryotic cells. Biochemical and cellular evidence show that HERMES employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. HERMES nucleases contain a re-arranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that HERMES can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Published
2023

28. RNA-activated protein cleavage with a CRISPR-associated endopeptidase

Author

Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Strecker, Jonathan, Demircioglu, F Esra, Li, David, Faure, Guilhem, Wilkinson, Max E, Gootenberg, Jonathan S, Abudayyeh, Omar O, Nishimasu, Hiroshi, Macrae, Rhiannon K, Zhang, Feng, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Strecker, Jonathan, Demircioglu, F Esra, Li, David, Faure, Guilhem, Wilkinson, Max E, Gootenberg, Jonathan S, Abudayyeh, Omar O, Nishimasu, Hiroshi, Macrae, Rhiannon K, and Zhang, Feng

Abstract

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

Published
2023

29. A transcription factor atlas of directed differentiation

Author

Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Joung, Julia, Ma, Sai, Tay, Tristan, Geiger-Schuller, Kathryn R, Kirchgatterer, Paul C, Verdine, Vanessa K, Guo, Baolin, Arias-Garcia, Mario A, Allen, William E, Singh, Ankita, Kuksenko, Olena, Abudayyeh, Omar O, Gootenberg, Jonathan S, Fu, Zhanyan, Macrae, Rhiannon K, Buenrostro, Jason D, Regev, Aviv, Zhang, Feng, Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences, Joung, Julia, Ma, Sai, Tay, Tristan, Geiger-Schuller, Kathryn R, Kirchgatterer, Paul C, Verdine, Vanessa K, Guo, Baolin, Arias-Garcia, Mario A, Allen, William E, Singh, Ankita, Kuksenko, Olena, Abudayyeh, Omar O, Gootenberg, Jonathan S, Fu, Zhanyan, Macrae, Rhiannon K, Buenrostro, Jason D, Regev, Aviv, and Zhang, Feng

Abstract

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.

Published
2023

34. RNA-activated protein cleavage with a CRISPR-associated endopeptidase

Author

Strecker, Jonathan, primary, Demircioglu, F. Esra, additional, Li, David, additional, Faure, Guilhem, additional, Wilkinson, Max E., additional, Gootenberg, Jonathan S., additional, Abudayyeh, Omar O., additional, Nishimasu, Hiroshi, additional, Macrae, Rhiannon K., additional, and Zhang, Feng, additional

Published
2022
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35. RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease

Author

Kato, Kazuki, primary, Okazaki, Sae, additional, Schmitt-Ulms, Cian, additional, Jiang, Kaiyi, additional, Zhou, Wenyuan, additional, Ishikawa, Junichiro, additional, Isayama, Yukari, additional, Adachi, Shungo, additional, Nishizawa, Tomohiro, additional, Makarova, Kira S., additional, Koonin, Eugene V., additional, Abudayyeh, Omar O., additional, Gootenberg, Jonathan S., additional, and Nishimasu, Hiroshi, additional

Published
2022
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36. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases

Author

Yarnall, Matthew T. N., primary, Ioannidi, Eleonora I., additional, Schmitt-Ulms, Cian, additional, Krajeski, Rohan N., additional, Lim, Justin, additional, Villiger, Lukas, additional, Zhou, Wenyuan, additional, Jiang, Kaiyi, additional, Garushyants, Sofya K., additional, Roberts, Nathaniel, additional, Zhang, Liyang, additional, Vakulskas, Christopher A., additional, Walker, John A., additional, Kadina, Anastasia P., additional, Zepeda, Adrianna E., additional, Holden, Kevin, additional, Ma, Hong, additional, Xie, Jun, additional, Gao, Guangping, additional, Foquet, Lander, additional, Bial, Greg, additional, Donnelly, Sara K., additional, Miyata, Yoshinari, additional, Radiloff, Daniel R., additional, Henderson, Jordana M., additional, Ujita, Andrew, additional, Abudayyeh, Omar O., additional, and Gootenberg, Jonathan S., additional

Published
2022
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37. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR

Author

Jiang, Kaiyi, primary, Koob, Jeremy, additional, Chen, Xi Dawn, additional, Krajeski, Rohan N., additional, Zhang, Yifan, additional, Volf, Verena, additional, Zhou, Wenyuan, additional, Sgrizzi, Samantha R., additional, Villiger, Lukas, additional, Gootenberg, Jonathan S., additional, Chen, Fei, additional, and Abudayyeh, Omar O., additional

Published
2022
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39. RNA-triggered protein cleavage and cell death by the RNA-guided type III-E CRISPR-Cas nuclease-protease complex

Author

Kato, Kazuki, primary, Okazaki, Sae, additional, Schmitt-Ulms, Cian, additional, Jiang, Kaiyi, additional, Zhou, Wenyuan, additional, Ishikawa, Junichiro, additional, Isayama, Yukari, additional, Adachi, Shungo, additional, Nishizawa, Tomohiro, additional, Makarova, Kira S., additional, Koonin, Eugene V., additional, Abudayyeh, Omar O., additional, Gootenberg, Jonathan S., additional, and Nishimasu, Hiroshi, additional

Published
2022
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41. A transcription factor atlas of directed differentiation

Author

Joung, Julia, Ma, Sai, Tay, Tristan, Geiger-Schuller, Kathryn R., Kirchgatterer, Paul C., Verdine, Vanessa K., Guo, Baolin, Arias-Garcia, Mario A., Allen, William E., Singh, Ankita, Kuksenko, Olena, Abudayyeh, Omar O., Gootenberg, Jonathan S., Fu, Zhanyan, Macrae, Rhiannon K., Buenrostro, Jason D., Regev, Aviv, and Zhang, Feng

Published
2024
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42. A Survey of Genome Editing Activity for 16 Cas12a Orthologs

Author

Broad Institute of MIT and Harvard, 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, Zetsche, Bernd, Abudayyeh, Omar O., Gootenberg, Jonathan S, Scott, David Arthur, Zhang, Feng, Broad Institute of MIT and Harvard, 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, Zetsche, Bernd, Abudayyeh, Omar O., Gootenberg, Jonathan S, Scott, David Arthur, and Zhang, Feng

Abstract

© 2019 by The Keio Journal of Medicine. The class 2 CRISPR-Cas endonuclease Cas12a (previously known as Cpf1) offers several advantages over Cas9, including the ability to process its own array and the requirement for just a single RNA guide. These attributes make Cas12a promising for many genome engineering applications. To further expand the suite of Cas12a tools available, we tested 16 Cas12a orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cas12a), exhibited robust indel formation. We also showed that Mb3Cas12a displays some tolerance for a shortened PAM (TTN versus the canonical Cas12a PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology., National Institutes of Health (Grants 1R01-HG009761, 1R01-MH110049 and 1DP1-HL141201)

Published
2022

43. Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13

Author

Broad Institute of MIT and Harvard, 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, Abudayyeh, Omar O., Gootenberg, Jonathan S, Kellner, Max J., Zhang, Feng, Broad Institute of MIT and Harvard, 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, Abudayyeh, Omar O., Gootenberg, Jonathan S, Kellner, Max J., and Zhang, Feng

Abstract

Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (∼15 min), quantitative, and portable, and can process crude soybean extracts as input material for minimal nucleic acid sample preparation. This field-ready SHERLOCK platform with color-based lateral flow readout can be applied for detection and quantitation of genes in a range of agricultural applications.

Published
2022

44. RNA targeting with CRISPR–Cas13

Author

Abudayyeh, Omar O., Gootenberg, Jonathan S., Essletzbichler, Patrick, Han, Shuo, Joung, Julia, Belanto, Joseph J., Verdine, Vanessa, Cox, David B. T., Kellner, Max J., Regev, Aviv, Lander, Eric S., Voytas, Daniel F., Ting, Alice Y., and Zhang, Feng

Published
2017
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47. A Survey of Genome Editing Activity for 16 Cas12a Orthologs

Author

Zetsche, Bernd, Strecker, Jonathan, Abudayyeh, Omar O., Gootenberg, Jonathan S., Scott, David A., and Zhang, Feng

Subjects
0301 basic medicine, CRISPR-Associated Proteins, Computational biology, Article, Genome engineering, 03 medical and health sciences, Endonuclease, 0302 clinical medicine, Bacterial Proteins, INDEL Mutation, Genome editing, Humans, Moraxella, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats, Indel, Base Pairing, Gene Editing, Endodeoxyribonucleases, Base Sequence, biology, Cas9, High-Throughput Nucleotide Sequencing, RNA, General Medicine, biology.organism_classification, Isoenzymes, HEK293 Cells, 030104 developmental biology, biology.protein, Nucleic Acid Conformation, Moraxella bovoculi, CRISPR-Cas Systems, Genetic Engineering, Sequence Alignment, 030217 neurology & neurosurgery, Plasmids, RNA, Guide, Kinetoplastida
Abstract

The class 2 CRISPR-Cas endonuclease Cas12a (previously known as Cpf1) offers several advantages over Cas9, including the ability to process its own array and the requirement for just a single RNA guide. These attributes make Cas12a promising for many genome engineering applications. To further expand the suite of Cas12a tools available, we tested 16 Cas12a orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cas12a), exhibited robust indel formation. We also showed that Mb3Cas12a displays some tolerance for a shortened PAM (TTN versus the canonical Cas12a PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.

Published
2020

48. Programmable eukaryotic protein expression with RNA sensors

Author

Jiang, Kaiyi, primary, Koob, Jeremy, additional, Chen, Xi Dawn, additional, Krajeski, Rohan N., additional, Zhang, Yifan, additional, Villiger, Lukas, additional, Zhou, Wenyuan, additional, Abudayyeh, Omar O., additional, Chen, Fei, additional, and Gootenberg, Jonathan S., additional

Published
2022
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49. Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

Author

Ioannidi, Eleonora I., primary, Yarnall, Matthew T. N., additional, Schmitt-Ulms, Cian, additional, Krajeski, Rohan N., additional, Lim, Justin, additional, Villiger, Lukas, additional, Zhou, Wenyuan, additional, Jiang, Kaiyi, additional, Roberts, Nathaniel, additional, Zhang, Liyang, additional, Vakulskas, Christopher A., additional, Walker, John A., additional, Kadina, Anastasia P., additional, Zepeda, Adrianna E., additional, Holden, Kevin, additional, Gootenberg, Jonathan S., additional, and Abudayyeh, Omar O., additional

Published
2021
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50. 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

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