Article
Agronomy
Xiaojing Liu, Jiangtao Yang, Yaya Song, Xiaochun Zhang, Xujing Wang, Zhixing Wang
Summary: CRISPR-Cas9 is a commonly used tool for gene editing. This study analyzed the impact of sgRNA length and number on editing efficiency in rice, finding that a 20 nt sgRNA was the most efficient. Additionally, transgenic rice with two sgRNAs showed higher editing efficiency compared to a single sgRNA.
Article
Biochemical Research Methods
Jianfeng Lei, Peihong Dai, Yue Li, Wanqi Zhang, Guantong Zhou, Chao Liu, Xiaodong Liu
Summary: The virus-induced genome editing (VIGE) system has great application potential, but it cannot transfer edited genes to the next generation due to the inability of viruses to enter shoot apical meristem (SAM) in plants. A novel CLCrV-mediated VIGE system using the FT strategy enables heritable mutant offspring without tissue culture in A. thaliana.
Article
Multidisciplinary Sciences
Junhong Choi, Wei Chen, Anna Minkina, Florence M. Chardon, Chase C. Suiter, Samuel G. Regalado, Silvia Domcke, Nobuhiko Hamazaki, Choli Lee, Beth Martin, Riza M. Daza, Jay Shendure
Summary: DNA Typewriter is a system for in vivo molecular recording that overcomes limitations of contemporary DNA-based memory devices. It utilizes insertional edits to record symbols and sequential genome editing to capture the order of events. It has the capability to record and decode complex events and short text messages, as well as potentially record multiple serial events.
Review
Cell Biology
Joseph Andrew Whitley, Houjian Cai
Summary: Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with great therapeutic potential. Extracellular vesicles (EVs) have emerged as a promising therapeutic delivery vehicle for CRISPR RNP encapsulation. Different strategies have been identified to encapsulate CRISPR RNP into EVs based on genetic fusion, protein tethering, and sgRNA-coupled encapsulation. Incorporating targeting moieties to EVs membrane surface can increase delivery efficiency. However, there are still limitations preventing the use of these strategies in clinical trials.
JOURNAL OF EXTRACELLULAR VESICLES
(2023)
Article
Biochemical Research Methods
Se Ra Lim, Ho Joung Lee, Hyun Ju Kim, Sang Jun Lee
Summary: Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for simultaneous and accurate editing of multiple genes. The use of truncated single-molecular guide RNA (sgRNA) method enables highly efficient editing at single-nucleotide resolution. This method holds significant potential for widespread and practical use in synthetic biology.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Microbiology
Zhiwei Hu, Yannan Wang, Qian Liu, Yan Qiu, Zhiyu Zhong, Kangdi Li, Wenhua Li, Zixin Deng, Yuhui Sun
Summary: This study introduces a novel approach using bubble hairpin single guide RNAs (BH-sgRNAs) to significantly reduce off-target editing and improve the purity of on-target editing in base editors. This design represents a major improvement in the specificity of base editing tools.
Review
Biotechnology & Applied Microbiology
Congting Guo, Xiaoteng Ma, Fei Gao, Yuxuan Guo
Summary: Gene editing involves precise changes to specific nucleic acid sequences. The CRISPR/Cas9 system has revolutionized gene editing, making it efficient, convenient, and programmable. However, off-target effects remain a major concern, leading to unexpected alterations in the genome. Various methods have been developed to detect and mitigate these off-target effects, advancing the precision of CRISPR/Cas9 derivatives. This review summarizes these technological advancements and discusses the challenges in managing off-target effects for future gene therapy.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2023)
Review
Genetics & Heredity
Chao Li, Wen Chu, Rafaqat Ali Gill, Shifei Sang, Yuqin Shi, Xuezhi Hu, Yuting Yang, Qamar U. Zaman, Baohong Zhang
Summary: In the past decade, significant progress has been made in developing versatile CRISPR/Cas nucleases and precise genome editors. Computer-assisted tools have played a crucial role in designing sgRNA and analyzing CRISPR/Cas-induced mutations. This review provides insights into the different features of CRISPR/Cas tools, the usage of computer-assisted tools, and the limitations and suggestions for further optimization.
GENOMICS PROTEOMICS & BIOINFORMATICS
(2023)
Review
Plant Sciences
Junming Zhou, Xinchao Luan, Yixuan Liu, Lixue Wang, Jiaxin Wang, Songnan Yang, Shuying Liu, Jun Zhang, Huijing Liu, Dan Yao
Summary: CRISPR-Cas9 gene editing technology is widely used for crop improvement due to its simplicity, high efficiency, specificity, and low production costs. Improving the gene-editing efficiency is crucial for molecular breeding, and this review discusses strategies such as Cas9 variant enzyme engineering, the use of multiple promoter-driven Cas9, and efficient optimization and expression of gRNA. It also briefly introduces optimization strategies for the CRISPR/Cas12a system and the application of precision editing.
Article
Biochemistry & Molecular Biology
Ho Joung Lee, Hyun Ju Kim, Sang Jun Lee
Summary: The study found that 5'-truncated sgRNA can be used for negatively selecting single-base edited targets in microbial genomes, as well as for simple and effective single-base editing, even with an engineered Cas9 nuclease with an expanded PAM.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Review
Biochemistry & Molecular Biology
Emre Aksoy, Kubilay Yildirim, Musa Kavas, Ceyhun Kayihan, Bayram Ali Yerlikaya, Irmak Calik, Ilkay Sevgen, Ufuk Demirel
Summary: CRISPR/Cas technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. It is more effective than conventional breeding methods or genetic engineering for developing climate resilient plant varieties. Knowledge in tissue culture-based plant transformation, effective sgRNA design, off-target prediction, and use of vectors, promoters, Cas proteins, and terminators is necessary. Various bioinformatics tools are available for sgRNA design and off-target screening.
MOLECULAR BIOLOGY REPORTS
(2022)
Article
Biotechnology & Applied Microbiology
Lukas Theo Schmitt, Aksana Schneider, Jonas Posorski, Felix Lansing, Milica Jelicic, Manavi Jain, Shady Sayed, Frank Buchholz, Duran Sueruen
Summary: DEQSeq is a nanopore sequencing approach that enables the rational selection of favorable genome editing enzymes from directed molecular evolution experiments. It streamlines the process of identifying valuable variants for further study and application by capturing full-length sequences, editing efficiencies, and specificities from thousands of evolved enzymes simultaneously.
Article
Biotechnology & Applied Microbiology
Ming-Zhi Cai, Po-Ting Chen
Summary: The Cre-Cas system combines Cre-lox72 and CRISPR-Cas9 to achieve genome editing efficiency in Bacillus subtilis through single homologous recombination, providing the feasibility of chromosomal editing in this bacterium.
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
(2021)
Article
Entomology
Xien Chen, Subba Reddy Palli
Summary: This study reports the establishment of multiple transgenic CRISPR/Cas9-based genome editing methods in the global agricultural pest, fall armyworm, Spodoptera frugiperda. Fluorescent proteins suitable for screening for transgenic fall armyworm were identified. The use of U6-tRNA-sgRNA system for sgRNA expression was found to be more efficient in inducing target gene knockout. These methods provide valuable tools for gene editing and functional genomics studies in the fall armyworm and other lepidopteran pests.
JOURNAL OF PEST SCIENCE
(2023)
Review
Microbiology
Yi Zeng, Yuxiang Hong, Fidelis Azi, Yugeng Liu, Yousheng Chen, Chuchu Guo, Dewei Lin, Zizhao Wu, Wenhao Chen, Peng Xu
Summary: This review discusses the development of efficient genome-editing tools and their application in generating large-scale genetic variants. These tools include traditional recombination-based tools, as well as more advanced CRISPR and RNA-based genome-editing tools. They have been used in strain engineering, biomanufacturing, and biosensing. Genome-editing technologies, along with high-throughput phenotyping and AI-based optimization algorithms, are expected to drive automation and streamline biological engineering, and help address critical challenges in health, environment, energy, and sustainability.
CURRENT OPINION IN MICROBIOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Nicholas Z. Lue, Emma M. Garcia, Kevin C. Ngan, Ceejay Lee, John G. Doench, Brian B. Liau
Summary: In this study, we use base editing and a DNA methylation reporter to scan mutations of DNMT3A in situ in cells. We identify mutations throughout the protein that disrupt function, including mutations at the interdomain interface that block allosteric activation. Surprisingly, we also find mutations in the PWWP domain that modulate enzyme activity by affecting DNA affinity.
NATURE CHEMICAL BIOLOGY
(2023)
Article
Multidisciplinary Sciences
Peter C. DeWeirdt, Abby V. McGee, Fengyi Zheng, Ifunanya Nwolah, Mudra Hegde, John G. Doench
Summary: This study develops a new on-target model that provides optimal predictions for multiple tracrRNA variants and demonstrates significant improvement over previous prediction models on a new dataset. By analyzing the differences in sgRNA activity between tracrRNA variants, the authors identify Pol III transcription termination as a strong determinant of sgRNA activity. These findings are expected to enhance the performance of CRISPR screening and inform future research on tracrRNA engineering and sgRNA modeling.
NATURE COMMUNICATIONS
(2022)
Article
Oncology
Kimberly R. Hagel, Rand Arafeh, Sydney Gang, Taylor E. Arnoff, Rebecca C. Larson, John G. Doench, Nathan D. Mathewson, Kai W. Wucherpfennig, Marcela Maus, William C. Hahn
Summary: This study uncovers multiple antigen-dependent and independent mechanisms of CAR T-cell evasion by pancreatic cancer, including the loss of genes involved in GPI-anchor biosynthesis and attachment pathway and genes that regulate tumor transcriptional responses. The identification of these resistance mechanisms provides insights into the landscape of tumor cell intrinsic resistance and potential approaches to improve CAR T-cell therapy efficacy in pancreatic cancer.
Article
Multidisciplinary Sciences
Matteo Gentili, Bingxu Liu, Malvina Papanastasiou, Deborah Dele-Oni, Marc A. Schwartz, Rebecca J. Carlson, Aziz M. Al'Khafaji, Karsten Krug, Adam Brown, John G. Doench, Steven A. Carr, Nir Hacohen
Summary: In this study, genes regulating STING trafficking were systematically characterized, and their impact on STING-mediated responses was examined. The study revealed that the ESCRT complex promotes STING degradation and terminates STING signaling. Furthermore, a UBAP1 mutation was found to increase steady-state STING-dependent responses. Overall, this study uncovers the role of the ESCRT complex in regulating STING signaling.
NATURE COMMUNICATIONS
(2023)
Article
Oncology
Yue Yan, Marc-Andre Gauthier, Ahmad Malik, Iosifina Fotiadou, Michael Ostrovski, Dzana Dervovic, Logine Ghadban, Ricky Tsai, Gerald Gish, Sampath Kumar Loganathan, Daniel Schramek
Summary: The aim of this study is to understand the signaling mechanisms of RIPK4 in SCCs. Through gene profiling and targeted CRISPR screening, we identified ELOVL4 as a downstream target of RIPK4. Our data from in vitro and in vivo mouse experiments show that the NOTCH-RIPK4-IRF6-ELOVL4 signaling axis functions as a potent tumor suppressor in SCCs.
Correction
Genetics & Heredity
Daniel Schramek, Athanassios Kotsinas, Arabella Meixner, Teiji Wada, Ulrich Elling, J. Andrew Pospisilik, G. Gregory Neely, Ralf-Harun Zwick, Verena Sigl, Guido Forni, Manuel Serrano, Vassilis G. Gorgoulis, Josef M. Penninger
Article
Oncology
Kathryn Gunn, Matti Myllykoski, John Z. Cao, Manna Ahmed, Bofu Huang, Betty Rouaisnel, Bill H. Diplass, Michael M. Levitt, Ryan Looper, John G. Doench, Keith L. Ligon, Harley I. Kornblum, Samuel K. McBrayers, Hai Yan, Cihangir Duy, Lucy A. Godley, Peppi Koivunen, Julie-Aurore Losman
Summary: Oncogenic mutations in IDH1 and IDH2 are commonly found in various cancers, including AML and glioma. The mutant IDH enzymes produce (R)-2HG, which is believed to promote cellular transformation by disrupting 2OG-dependent enzymes. This study reveals that (R)-2HG inhibits KDM5 histone lysine demethylases, contributing to cellular transformation in IDH-mutant AML and glioma.
Article
Oncology
Yoshinaga Ito, Deng Pan, Wubing Zhang, Xixi Zhang, Tiffany Y. Juan, Jason W. Pyrdol, Oleksandr Kyrysyuk, John G. Doench, X. Shirley Liu, Kai W. Wucherpfennig
Summary: Tumor heterogeneity is a major obstacle in cancer therapy, including immunotherapy. By targeting autophagy and TNF signaling pathways, MHC-I-deficient tumor cells can be sensitized to T cell-mediated apoptosis. Antigens from apoptotic MHC-I-deficient tumor cells can be efficiently cross-presented by dendritic cells, leading to increased infiltration of T cells producing IFN-γ and TNF-α. Targeting both pathways enables T cell-mediated elimination of tumors with a substantial population of resistant, MHC-I-deficient cells.
Article
Medicine, Research & Experimental
Tianxia Li, Osamu Kikuchi, Jin Zhou, Yichen Wang, Babita Pokharel, Klavdija Bastl, Prafulla Gokhale, Aine Knott, Yanxi Zhang, John G. Doench, Zandra V. Ho, Daniel V. T. Catenacci, Adam J. Bass
Summary: Gastroesophageal adenocarcinomas (GEAs) frequently exhibit amplification of KRAS gene, resulting in overexpression of WT KRAS protein. This study investigates potential targets to enhance the efficacy of SHP2 inhibition in KRAS-amplified GEA, including those within the MAPK pathway and upstream RTKs. In vitro and in vivo experiments demonstrate that pan-ERBB kinase inhibition has potent cytotoxicity. Additionally, the combination of CDK4/6 inhibition with SHP2 inhibition shows greater efficacy in KRAS-amplified GEA compared to KRAS-mutant tumors. These findings suggest potential therapeutic combinations for clinical study in KRAS-amplified GEAs.
Article
Oncology
Ana Vujovic, Laura de Rooij, Ava Keyvani Chahi, He Tian Chen, Brian A. Yee, Sampath K. Loganathan, Lina Liu, Derek C. H. Chan, Amanda Tajik, Emily Tsao, Steven Moreira, Pratik Joshi, Joshua Xu, Nicholas Wong, Zaldy Balde, Soheil Jahangiri, Sasan Zandi, Stefan Aigner, John E. Dick, Mark D. Minden, Daniel Schramek, Gene W. Yeo, Kristin J. Hope
Summary: Acute myeloid leukemia (AML) is driven by leukemic stem cells (LSC) and identifying the determinants of LSCs is challenging. This study identified a group of RNA-binding proteins (RBPs) enriched in human AML LSCs. Through a stem cell-adapted CRISPR screen, they found 32 essential RBPs for LSCs and discovered that ELAVL1, an RBP, plays a crucial role in AML propagation by regulating mitochondrial metabolism. These findings highlight the importance of RBPs as therapeutic targets in AML.
BLOOD CANCER DISCOVERY
(2023)
Article
Multidisciplinary Sciences
Gintautas Vainorius, Maria Novatchkova, Georg Michlits, Juliane Christina Baar, Cecilia Raupach, Joonsun Lee, Ramesh Yelagandula, Marius Wernig, Ulrich Elling
Summary: In this study, the authors show that cells can take two mechanistically distinct paths in the same transition paradigm when driven by the similar proneural factors Ascl1 and Ngn2. Ascl1 dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network.
NATURE COMMUNICATIONS
(2023)
Review
Immunology
Hao Shi, John G. Doench, Hongbo Chi
Summary: CRISPR-based technologies offer a powerful platform for unbiased screening and functional genomics in various fields, including immunology. These technologies have uncovered previously unknown intracellular drivers and intercellular regulators in immune cells, and have expanded the readouts to the transcriptome for better mechanistic insights. CRISPR screens also allow for mapping of genetic interactions to identify genes that synergize or alleviate complex immune phenotypes.
NATURE REVIEWS IMMUNOLOGY
(2023)
Meeting Abstract
Ophthalmology
Karina Luiza Dias Teixeira, Mazhar Adli, John Doench, Gordon W. Laurie
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
(2022)
Meeting Abstract
Oncology
Jessica W. Tsai, Frank P. B. Dubois, Dayle K. Wang, Alexander Crane, Alexandra L. Condurat, Brian Krug, Adam Brown, John G. Doench, Nada Jabado, Pratiti Bandopadhayay
