Review
Pharmacology & Pharmacy
Diana Campillo-Davo, Maxime De Laere, Gils Roex, Maarten Versteven, Donovan Flumens, Zwi N. Berneman, Viggo F. I. Van Tendeloo, Sebastien Anguille, Eva Lion
Summary: Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells that allows fine-tuning of transfection protocols for each cell type and introduction of multiple protein-coding mRNAs at once. In this review, the parameters involved in mRNA electroporation and the production and application of clinical-grade mRNA for gene transfer in cell-based immunotherapies are discussed by a pioneering group in mRNA electroporation.
Editorial Material
Biochemistry & Molecular Biology
Jean M. Kanellopoulos, David M. Ojcius
Summary: This special edition provides a summary of significant progress in understanding signaling by T lymphocytes. It explores the changes in T cell adhesion and actin cytoskeleton during interactions with antigen-presenting cells (APCs) and other immune cells. The articles discuss the mediators of these changes within T cells and on the T cell surface. They specifically focus on inside-out integrin signaling, components of the immunological synapse, T cell receptor signaling from endosomes, membrane transfer between APCs and T cells via trogocytosis, immune deficiencies in T cell signaling pathways, and the role of THEMIS in thymocyte development and peripheral T cell function.
BIOMEDICAL JOURNAL
(2022)
Article
Biotechnology & Applied Microbiology
Philipp Reautschnig, Nicolai Wahn, Jacqueline Wettengel, Annika E. Schulz, Ngadhnjim Latifi, Paul Vogel, Tae-Won Kang, Laura S. Pfeiffer, Christine Zarges, Ulrike Naumann, Lars Zender, Jin Billy Li, Thorsten Stafforst
Summary: RNA base editing is a promising alternative to genome editing, and the use of CLUSTER guide RNAs can enhance editing efficiency and precision. The multivalent binding of CLUSTER gRNAs to target messenger RNAs enables editing in various cell lines without bystander effects.
NATURE BIOTECHNOLOGY
(2022)
Review
Cell Biology
Paula Germino-Watnick, Malikiya Hinds, Anh Le, Rebecca Chu, Xiong Liu, Naoya Uchida
Summary: Autologous hematopoietic stem cell (HSC)-targeted gene therapy provides a one-time cure for various genetic diseases including sickle cell disease (SCD) and beta-thalassemia. This review discusses the methods of gene addition and gene editing in HSC-targeted gene therapy for SCD.
Review
Biochemistry & Molecular Biology
Dito Anurogo, Nova Yuli Prasetyo Budi, Mai-Huong Thi Ngo, Yen-Hua Huang, Jeanne Adiwinata Pawitan
Summary: Hereditary anemia presents with various manifestations, and current management strategies are unsatisfactory. Gene-corrected hematopoietic stem cell transplantation may offer promising outcomes.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Editorial Material
Microbiology
Rajendra Prasad, Atanu Banerjee
Summary: The authors have created a double gene deletion library using yeast genetics, which allows high-throughput identification of drug and xenobiotic transporters, addressing the challenges of substrate promiscuity and functional redundancy. They found a strong correlation between the chemical structure of azoles and possible import/export routes, and identified specific transporters responsible for the import of certain compounds.
Review
Biochemistry & Molecular Biology
Christelle Gross, Louis-Philippe Guerin, Bianca G. Socol, Lucie Germain, Sylvain L. Guerin
Summary: CLU is a glycoprotein that is widely distributed in human tissues and plays important roles in tissue repair and cellular function. Its expression is increased in certain eye diseases, but its precise role needs further investigation.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Multidisciplinary Sciences
Gregory A. Newby, Jonathan S. Yen, Kaitly J. Woodard, Thiyagaraj Mayuranathan, Cicera R. Lazzarotto, Yichao Li, Heather Sheppard-Tillman, Shaina N. Porter, Yu Yao, Kalin Mayberry, Kelcee A. Everette, Yoonjeong Jang, Christopher J. Podracky, Elizabeth Thaman, Christophe Lechauve, Akshay Sharma, Jordana M. Henderson, Michelle F. Richter, Kevin T. Zhao, Shannon M. Miller, Tina Wang, Luke W. Koblan, Anton P. McCaffrey, John F. Tisdale, Theodosia A. Kalfa, Shondra M. Pruett-Miller, Shengdar Q. Tsai, Mitchell J. Weiss, David R. Liu
Summary: The study demonstrated successful conversion of sickle cell disease allele into a non-pathogenic variant using adenine base editor, with durable therapeutic effects. The edited HSPCs improved physiological parameters and reduced pathological abnormalities in spleens of mice, indicating the potential for long-lasting and effective treatment for SCD.
Review
Biochemistry & Molecular Biology
Maria Agnese Della-Fazia, Marilena Castelli, Danilo Piobbico, Stefania Pieroni, Giuseppe Servillo
Summary: HOPS plays a crucial role in liver regeneration by contributing to genomic stability, controlling cell cycle, and interacting with other key proteins.
Article
Chemistry, Multidisciplinary
Lei Qiao, Min Gao, Xiaoqing Yi, Hui Peng, Ruijie Zhang, Wanqing Yao, Gengyun Sun, Xiaoyan He
Summary: This study developed a cell membrane biomimetic core-shell system for light-controllable, precise gene editing, and successfully applied it in inhibiting tumor metastasis and enhancing chemotherapy effects. The system consisted of a core for CRISPR-Cas9/sgRNA plasmid loading and a shell camouflaged by a cell membrane, which induced lysosomal escape and payload release through reactive oxygen species production. By using this system, the expression of VEGF and Vimentin in H1299 tumor cells decreased, leading to enhanced antimetastatic effects. The results demonstrate that the membrane-camouflaged system combined with light augmentation provides a potential solution for in vivo delivery of CRISPR-Cas9 and cancer therapy.
JOURNAL OF CONTROLLED RELEASE
(2023)
Review
Physiology
Oscar Quintana-Bustamante, Sara Fananas-Baquero, Mercedes Dessy-Rodriguez, Isabel Ojeda-Perez, Jose-Carlos Segovia
Summary: Gene therapy is a viable option for treating inherited hematological diseases, and gene editing techniques, such as CRISPR/Cas9, offer precise and safe approaches for correcting genetic mutations. Promising results have been seen in the treatment of red blood cell diseases, and the field is rapidly moving towards clinical applications.
FRONTIERS IN PHYSIOLOGY
(2022)
Review
Medicine, General & Internal
Ella S. Atsavapranee, Margaret M. Billingsley, Michael J. Mitchell
Summary: Genetic engineering has transformed cancer immunotherapy by modifying primary T cells to enhance their therapeutic potential, with studies and clinical trials supporting the effectiveness of this approach.
Article
Multidisciplinary Sciences
John A. Morris, Christina Caragine, Zharko Daniloski, Julia Domingo, Timothy Barry, Lu Lu, Kyrie Davis, Marcello Ziosi, Dafni A. Glinos, Stephanie Hao, Eleni P. Mimitou, Peter Smibert, Kathryn Roeder, Eugene Katsevich, Tuuli Lappalainen, Neville E. Sanjana
Summary: By using ancestrally diverse, biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we have identified 124 cis target genes of 91 noncoding blood trait GWAS loci. Through precise variant insertion via base editing, we have connected specific variants with gene expression changes. We have also discovered trans-effect networks of noncoding loci when cis target genes encode transcription factors or microRNAs.
Article
Multidisciplinary Sciences
Mathilde Beaufils, Amandine Tourel, Anne Petiot, Nicole B. Halmai, Dave J. Segal, John Rendu, Isabelle Marty
Summary: One important application of CRISPR/Cas9 is the development of knock-out cell lines to study the function of disease-associated genes/proteins. This protocol describes the insertion of CRISPR tools into difficult to transfect cells, such as muscle cells, using lentiviruses. The control of Cas9 activity allows the development of a RYR1-knockout human muscle cell line for further characterization.
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
(2022)
Article
Biochemistry & Molecular Biology
Rui Tao, Yanhong Wang, Yun Hu, Yaoge Jiao, Lifang Zhou, Lurong Jiang, Li Li, Xingyu He, Min Li, Yamei Yu, Qiang Chen, Shaohua Yao
Summary: A novel prime editor, WT-PE, was designed in this study by fusing reverse transcriptase (RT) to nuclease wild-type Cas9, enabling efficient editing of large genomic fragments. The WT-PE system introduced a double strand break (DSB) and a single 3' extended flap in the target site, and coupled with paired prime editing guide RNAs (pegRNAs), it achieved bi-directional prime editing, allowing versatile and efficient large-scale genome editing. This WT-PE system has great potential for modeling or treating diseases related to large-fragment aberrations.
SIGNAL TRANSDUCTION AND TARGETED THERAPY
(2022)
Article
Biochemistry & Molecular Biology
Theodore L. Roth, P. Jonathan Li, Franziska Blaeschke, Jasper F. Nies, Ryan Apathy, Cody Mowery, Ruby Yu, Michelle L. T. Nguyen, Youjin Lee, Anna Truong, Joseph Hiatt, David Wu, David N. Nguyen, Daniel Goodman, Jeffrey A. Bluestone, Chun Jimmie Ye, Kole Roybal, Eric Shifrut, Alexander Marson
Article
Multidisciplinary Sciences
Jessica T. Cortez, Elena Montauti, Eric Shifrut, Jovylyn Gatchalian, Yusi Zhang, Oren Shaked, Yuanming Xu, Theodore L. Roth, Dimitre R. Simeonov, Yana Zhang, Siqi Chen, Zhongmei Li, Jonathan M. Woo, Josephine Ho, Ian A. Vogel, Grace Y. Prator, Bin Zhang, Youjin Lee, Zhaolin Sun, Igal Ifergan, Frederic Van Gool, Diana C. Hargreaves, Jeffrey A. Bluestone, Alexander Marson, Deyu Fang
Article
Multidisciplinary Sciences
David E. Gordon, Gwendolyn M. Jang, Mehdi Bouhaddou, Jiewei Xu, Kirsten Obernier, Kris M. White, Matthew J. O'Meara, Veronica V. Rezelj, Jeffrey Z. Guo, Danielle L. Swaney, Tia A. Tummino, Ruth Huttenhain, Robyn M. Kaake, Alicia L. Richards, Beril Tutuncuoglu, Helene Foussard, Jyoti Batra, Kelsey Haas, Maya Modak, Minkyu Kim, Paige Haas, Benjamin J. Polacco, Hannes Braberg, Jacqueline M. Fabius, Manon Eckhardt, Margaret Soucheray, Melanie J. Bennett, Merve Cakir, Michael J. McGregor, Qiongyu Li, Bjoern Meyer, Ferdinand Roesch, Thomas Vallet, Alice Mac Kain, Lisa Miorin, Elena Moreno, Zun Zar Chi Naing, Yuan Zhou, Shiming Peng, Ying Shi, Ziyang Zhang, Wenqi Shen, Ilsa T. Kirby, James E. Melnyk, John S. Chorba, Kevin Lou, Shizhong A. Dai, Inigo Barrio-Hernandez, Danish Memon, Claudia Hernandez-Armenta, Jiankun Lyu, Christopher J. P. Mathy, Tina Perica, Kala Bharath Pilla, Sai J. Ganesan, Daniel J. Saltzberg, Ramachandran Rakesh, Xi Liu, Sara B. Rosenthal, Lorenzo Calviello, Srivats Venkataramanan, Jose Liboy-Lugo, Yizhu Lin, Xi-Ping Huang, YongFeng Liu, Stephanie A. Wankowicz, Markus Bohn, Maliheh Safari, Fatima S. Ugur, Cassandra Koh, Nastaran Sadat Savar, Quang Dinh Tran, Djoshkun Shengjuler, Sabrina J. Fletcher, Michael C. O'Neal, Yiming Cai, Jason C. J. Chang, David J. Broadhurst, Saker Klippsten, Phillip P. Sharp, Nicole A. Wenzell, Duygu Kuzuoglu-Ozturk, Hao-Yuan Wang, Raphael Trenker, Janet M. Young, Devin A. Cavero, Joseph Hiatt, Theodore L. Roth, Ujjwal Rathore, Advait Subramanian, Julia Noack, Mathieu Hubert, Robert M. Stroud, Alan D. Frankel, Oren S. Rosenberg, Kliment A. Verba, David A. Agard, Melanie Ott, Michael Emerman, Natalia Jura, Mark von Zastrow, Eric Verdin, Alan Ashworth, Olivier Schwartz, Christophe D'Enfert, Shaeri Mukherjee, Matt Jacobson, Harmit S. Malik, Danica G. Fujimori, Trey Ideker, Charles S. Craik, Stephen N. Floor, James S. Fraser, John D. Gross, Andrej Sali, Bryan L. Roth, Davide Ruggero, Jack Taunton, Tanja Kortemme, Pedro Beltrao, Marco Vignuzzi, Adolfo Garcia-Sastre, Kevan M. Shokat, Brian K. Shoichet, Nevan J. Krogan
Review
Medicine, Research & Experimental
Theodore L. Roth, Alexander Marson
Summary: Genetic diseases lead to complex and difficult pathologies, with DNA sequences containing disease risks in various genetic compartments. Current diagnosis strategies focus on next-generation DNA sequencing, while targeted genetic therapies aim at replacing, adding, or editing genetic material. Developing universal methods for diagnosis, therapy, and reagent delivery into genetic compartments will accelerate the advancement of curative genetic therapies.
ANNUAL REVIEW OF PATHOLOGY: MECHANISMS OF DISEASE, VOL 16, 2021
(2021)
Article
Medicine, Research & Experimental
Rachel L. Rutishauser, Christian Deo T. Deguit, Joseph Hiatt, Franziska Blaeschke, Theodore L. Roth, Lynn Wang, Kyle A. Raymond, Carly E. Starke, Joseph C. Mudd, Wenxuan Chen, Carolyn Smullin, Rodrigo Matus-Nicodemos, Rebecca Hoh, Melissa Krone, Frederick M. Hecht, Christopher D. Pilcher, Jeffrey N. Martin, Richard A. Koup, Daniel C. Douek, Jason M. Brenchley, Rafick-Pierre Sekaly, Satish K. Pillai, Alexander Marson, Steven G. Deeks, Joseph M. McCune, Peter W. Hunt
Summary: The transcription factor TCF-1 plays a crucial role in regulating the memory properties and expansion capacity of virus-specific CD8(+) T cells, which are important for controlling HIV infection. Increasing TCF-1 levels may enhance the secondary expansion capacity of HIV-specific CD8(+) T cells.
Article
Medicine, Research & Experimental
Sana Siddiqui, Kristina Johansson, Alex Joo, Luke R. Bonser, Kyung Duk Koh, Olivier Le Tonqueze, Samaneh Bolourchi, Rodriel A. Bautista, Lorna Zlock, Theodore L. Roth, Alexander Marson, Nirav R. Bhakta, K. Mark Ansel, Walter E. Finkbeiner, David J. Erle, Prescott G. Woodruff
Summary: The study identifies miR-141 as a key regulator of pathological airway mucus production induced by IL-13, with disruption leading to decreased goblet cell frequency and reduced mucus secretion. This miRNA may offer a novel therapeutic target for airway diseases.
Article
Cell Biology
Joseph Hiatt, Devin A. Cavero, Michael J. McGregor, Weihao Zheng, Jonathan M. Budzik, Theodore L. Roth, Kelsey M. Haas, David Wu, Ujjwal Rathore, Anke Meyer-Franke, Mohamed S. Bouzidi, Eric Shifrut, Youjin Lee, Vigneshwari Easwar Kumar, Eric Dang, David E. Gordon, Jason A. Wojcechowskyj, Judd F. Hultquist, Krystal A. Fontaine, Satish K. Pillai, Jeffery S. Cox, Joel D. Ernst, Nevan J. Krogan, Alexander Marson
Summary: A method for delivering CRISPR-Cas9 ribonucleoprotein complexes via nucleofection into human monocytes has been developed for precise gene knockout and differentiation into macrophages or dendritic cells. This system shows promise for genetic studies of human myeloid cells in various areas such as immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond.
Article
Immunology
Yannick D. Muller, Duy P. Nguyen, Leonardo M. R. Ferreira, Patrick Ho, Caroline Raffin, Roxxana Valeria Beltran Valencia, Zion Congrave-Wilson, Theodore L. Roth, Justin Eyquem, Frederic Van Gool, Alexander Marson, Laurent Perez, James A. Wells, Jeffrey A. Bluestone, Qizhi Tang
Summary: This study revealed a fundamental difference between CD28-TMD and CD8-TMD, indicating that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.
FRONTIERS IN IMMUNOLOGY
(2021)
Article
Multidisciplinary Sciences
Youjin Lee, Derek Bogdanoff, Yutong Wang, George C. Hartoularos, Jonathan M. Woo, Cody T. Mowery, Hunter M. Nisonoff, David S. Lee, Yang Sun, James Lee, Sadaf Mehdizadeh, Joshua Cantlon, Eric Shifrut, David N. Ngyuen, Theodore L. Roth, Yun S. Song, Alexander Marson, Eric D. Chow, Chun Jimmie Ye
Summary: Using the XYZeq workflow, spatial metadata can be encoded into scRNAseq libraries to analyze the transcriptome and spatial localization of individual cells, revealing the functional interactions within an anatomical space. Studies show that tumor-associated MSCs express different tumor suppressor genes at varying distances from the tumor core, highlighting the spatial distribution and functional characteristics of cells in pathological tissues.
Article
Biology
Dasmanthie De Silva, Lucas Ferguson, Grant H. Chin, Benjamin E. Smith, Ryan A. Apathy, Theodore L. Roth, Franziska Blaeschke, Marek Kudla, Alexander Marson, Nicholas T. Ingolia, Jamie H. D. Cate
Summary: This study reveals that human translation initiation factor eIF3 interacts with specific immune-related mRNAs and regulates T cell receptor translation, enhancing T cell activation. Utilizing this mechanism for genetic engineering improves the efficacy of CAR-T cells in killing tumor cells.
Article
Multidisciplinary Sciences
Joseph Hiatt, Judd F. Hultquist, Michael J. McGregor, Mehdi Bouhaddou, Ryan T. Leenay, Lacy M. Simons, Janet M. Young, Paige Haas, Theodore L. Roth, Victoria Tobin, Jason A. Wojcechowskyj, Jonathan M. Woo, Ujjwal Rathore, Devin A. Cavero, Eric Shifrut, Thong T. Nguyen, Kelsey M. Haas, Harmit S. Malik, Jennifer A. Doudna, Andrew P. May, Alexander Marson, Nevan J. Krogan
Summary: This study systematically evaluates the functional roles of 426 genes in HIV replication by using CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells. They identify 86 candidate host factors that alter HIV infection, providing new insights into HIV biology.
NATURE COMMUNICATIONS
(2022)
Article
Biotechnology & Applied Microbiology
Brian R. Shy, Vivasvan S. Vykunta, Alvin Ha, Alexis Talbot, Theodore L. Roth, David N. Nguyen, Wolfgang G. Pfeifer, Yan Yi Chen, Franziska Blaeschke, Eric Shifrut, Shane Vedova, Murad R. Mamedov, Jing-Yi Jing Chung, Hong Li, Ruby Yu, David Wu, Jeffrey Wolf, Thomas G. Martin, Carlos E. Castro, Lumeng Ye, Jonathan H. Esensten, Justin Eyquem, Alexander Marson
Summary: By using single-stranded DNA HDR templates with reduced toxicity and small-molecule combinations, the efficiency of CRISPR-mediated site-specific transgene insertion can be greatly enhanced. This method is applicable to various target loci, knock-in constructs, and primary human cell types, with high HDR efficiencies. Furthermore, it can be applied in pathogenic gene variant modeling, gene-replacement strategies, and nonviral chimeric antigen receptor-T cell manufacturing with improved knock-in efficiencies and yields.
NATURE BIOTECHNOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Franziska Blaeschke, Yan Yi Chen, Ryan Apathy, Bence Daniel, Andy Y. Chen, Peixin Amy Chen, Katalin Sandor, Wenxi Zhang, Zhongmei Li, Cody T. Mowery, Tori N. Yamamoto, William A. Nyberg, Angela To, Ruby Yu, Raymund Bueno, Min Cheol Kim, Ralf Schmidt, Daniel B. Goodman, Tobias Feuchtinger, Justin Eyquem, Chun Jimmie, Julia Carnevale, Ansuman T. Satpathy, Eric Shifrut, Theodore L. Roth, Alexander Marson
Summary: Chronic stimulation can lead to T cell dysfunction, limiting the effectiveness of cellular immunotherapies. Improved methods are needed to compare synthetic knockin sequences and reprogram cell functions. Modular pooled knockin screening was developed, allowing the construction of DNA libraries that identified a transcription factor TFAP4, which enhanced the fitness and anti-cancer function of CAR-T cells. The modularity of the platform facilitated the discovery of complex gene constructs to program cellular functions.
Article
Multidisciplinary Sciences
Julia Carnevale, Eric Shifrut, Nupura Kale, William A. Nyberg, Franziska Blaeschke, Yan Yi Chen, Zhongmei Li, Sagar P. Bapat, Morgan E. Diolaiti, Patrick O'Leary, Shane Vedova, Julia Belk, Bence Daniel, Theodore L. Roth, Stefanie Bachl, Alejandro Allo Anido, Brooke Prinzing, Jorge Ibanez-Vega, Shannon Lange, Dalia Haydar, Marie Luetke-Eversloh, Maelys Born-Bony, Bindu Hegde, Scott Kogan, Tobias Feuchtinger, Hideho Okada, Ansuman T. Satpathy, Kevin Shannon, Stephen Gottschalk, Justin Eyquem, Giedre Krenciute, Alan Ashworth, Alexander Marson
Summary: This study identifies RASA2 as a potential target gene that can enhance the efficacy of T cell therapies for cancer treatment. Ablation of RASA2 increases the activation and cytolytic activity of T cells, leading to improved cancer cell killing. Furthermore, RASA2-deficient CAR T cells have a competitive advantage in the bone marrow and extended the survival of mice in preclinical models. These findings highlight the importance of targeting RASA2 in enhancing T cell therapies for cancer treatment.