Article
Biotechnology & Applied Microbiology
David M. Ichikawa, Osama Abdin, Nader Alerasool, Manjunatha Kogenaru, April L. Mueller, Han Wen, David O. Giganti, Gregory W. Goldberg, Samantha Adams, Jeffrey M. Spencer, Rozita Razavi, Satra Nim, Hong Zheng, Courtney Gionco, Finnegan T. Clark, Alexey Strokach, Timothy R. Hughes, Timothee Lionnet, Mikko Taipale, Philip M. Kim, Marcus B. Noyes
Summary: ZFDesign, a deep-learning model, is developed to facilitate the design of Cys(2)His(2) zinc finger (ZF) domains for programmable regulation of gene expression. The model screens billions of protein-DNA interactions and provides a solution for the challenging process of ZF design. The designed ZFs demonstrate versatility as nucleases, activators, and repressors.
NATURE BIOTECHNOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Kathleen A. Christie, Jimmy A. Guo, Rachel A. Silverstein, Roman M. Doll, Megumu Mabuchi, Hannah E. Stutzman, Jiecong Lin, Linyuan Ma, Russell T. Walton, Luca Pinello, G. Brett Robb, Benjamin P. Kleinstiver
Summary: In this study, the utility of a near-PAMless SpCas9 variant named SpRY as a universal DNA cleavage tool for various cloning applications was explored. It was found that SpRY can cleave DNA in vitro without the need for a PAM, and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. The versatility and effectiveness of SpRYgests were illustrated to improve the precision of several cloning workflows, and a rapid and simple one-pot gRNA synthesis protocol was optimized to streamline SpRYgest implementation.
NATURE BIOTECHNOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Jiacheng Hu, Yu Sun, Boshu Li, Zhen Liu, Zhiwei Wang, Qiang Gao, Mengyue Guo, Guanwen Liu, Kevin Tianmeng Zhao, Caixia Gao
Summary: CyDENT is a CRISPR-free modular base editing tool with strand selectivity. It utilizes TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase, and an exonuclease to achieve efficient base editing in nuclear, mitochondrial, and chloroplast genomes.
NATURE BIOTECHNOLOGY
(2023)
Article
Multidisciplinary Sciences
Yanbo Wang, John Mallon, Haobo Wang, Digvijay Singh, Myung Hyun Jo, Boyang Hua, Scott Bailey, Taekjip Ha
Summary: The research discovered that the HNH domain of CRISPR-Cas9 exhibits high flexibility post-DNA cleavage, exposing a 3' flap after cleavage of the non-target strand, potentially interacting with the DNA repair machinery. These findings reveal the regulatory role of DNA cleavage activity on Cas9 conformation and suggest potential applications in biotechnology.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Biochemistry & Molecular Biology
Yanbo Wang, Wayne Taylor Cottle, Haobo Wang, Xinyu Ashlee Feng, John Mallon, Momcilo Gavrilov, Scott Bailey, Taekjip Ha
Summary: The study presents a new DNA detection method called GOLD FISH, utilizing Cas9 in complex with programmable guide RNA and superhelicase to label genomic DNA, achieving reduced nonspecific binding and avoiding harsh heat denaturation while maintaining signal brightness and specificity.
Article
Multidisciplinary Sciences
Lin Zhao, Sabrina R. T. Koseki, Rachel A. Silverstein, Nadia Amrani, Christina Peng, Christian Kramme, Natasha Savic, Martin Pacesa, Tomas C. Rodriguez, Teodora Stan, Emma Tysinger, Lauren Hong, Vivian Yudistyra, Manvitha R. Ponnapati, Joseph M. Jacobson, George M. Church, Noah Jakimo, Ray Truant, Martin Jinek, Benjamin P. Kleinstiver, Erik J. Sontheimer, Pranam Chatterjee
Summary: In this study, a chimeric enzyme SpRYc with highly flexible PAM preference was generated by combining the PAM-interacting domain of SpRY and the N-terminus of Sc++, leveraging properties of both enzymes. SpRYc showed the ability to specifically edit diverse PAMs and disease-related loci, highlighting the power of integrative protein design for Cas9 engineering.
NATURE COMMUNICATIONS
(2023)
Article
Chemistry, Multidisciplinary
Mengyi Yang, Ruirui Sun, Pujuan Deng, Yuzhuo Yang, Wenjuan Wang, Jun-Jie Gogo Liu, Chunlai Chen
Summary: Research has revealed the unique searching mechanism of Cas9 under physiological salt conditions, involving both three-dimensional and one-dimensional diffusion, as well as the presence of apparent asymmetric target search regions flanking PAM sites.
Article
Biochemistry & Molecular Biology
So Young Bak, Youngri Jung, Jinho Park, Keewon Sung, Hyeon-Ki Jang, Sangsu Bae, Seong Keun Kim
Summary: This study utilized single-molecule FRET assay to investigate the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants, revealing a strong correlation between their conformational structure and nuclease activity. Quantitative analysis showed that these engineered Cas9 variants exhibit a more rapid decrease in cleavage-competent state with increasing base-pair mismatch, leading to enhanced target specificity. The critical specificity enhancement step was found to be activated during DNA:RNA heteroduplexation for some variants, highlighting the importance of conformational dynamics in Cas9 target specificity.
NUCLEIC ACIDS RESEARCH
(2021)
Article
Multidisciplinary Sciences
Zhaohui Zhong, Guanqing Liu, Zhongjie Tang, Shuyue Xiang, Liang Yang, Lan Huang, Yao He, Tingting Fan, Shishi Liu, Xuelian Zheng, Tao Zhang, Yiping Qi, Jian Huang, Yong Zhang
Summary: In this study, a probiotic sourced CRISPR-LrCas9 system with a similar PAM requirement to Cas12a was reported, and its high efficiency in various genome editing applications was demonstrated.
NATURE COMMUNICATIONS
(2023)
Article
Multidisciplinary Sciences
Martin Pacesa, Luuk Loeff, Irma Querques, Lena M. Muckenfuss, Marta Sawicka, Martin Jinek
Summary: Researchers have used cryo-electron microscopy to observe the structural changes that occur during the binding and activation of Cas9. These findings provide a valuable structural framework for understanding the conformational checkpoint mechanism of Cas9 and may guide the development of new Cas9 variants and guide RNA designs with enhanced specificity and activity.
Article
Biochemistry & Molecular Biology
Ya-Jing Shi, Min Duan, Jun-Mei Ding, Fan-Qi Wang, Li-Li Bi, Cai-Xiang Zhang, Yi-Zhou Zhang, Jun-Yi Duan, An-Hui Huang, Xin-Lin Lei, Hao Yin, Ying Zhang
Summary: A Cas9 protein (Alicyclobacillus tengchongensis) from a thermophilic bacterium has been found to regulate PAM recognition through DNA topology. Structural analysis reveals that AtCas9 has a unique loop motif that interacts with the DNA major groove to modulate PAM recognition. Experimental results demonstrate the near-PAMless editing capability of AtCas9 in E. coli and its broad PAM preference for efficient editing in mammalian cells.
Article
Multidisciplinary Sciences
Dusko Lainscek, Vida Forstneric, Veronika Mikolic, Spela Malensek, Peter Pecan, Mojca Bencina, Matjaz Sever, Helena Podgornik, Roman Jerala
Summary: The CRISPR/Cas system, when coupled with Exonuclease III, can enhance gene editing efficiency and lead to significant gene knock-out effects, especially in cell lines, primary cells, and in vivo. This approach has great potential for treating diseases with a genetic etiology.
NATURE COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Aakash Saha, Pablo R. Arantes, Giulia Palermo
Summary: This article reviews the role of molecular simulations in the CRISPR-Cas9 revolution, including studies on RNA binding, catalytic mechanism, off-target effects, etc. It reveals the dynamics and mechanism of CRISPR-Cas9, contributing to understanding its function and specificity.
CURRENT OPINION IN STRUCTURAL BIOLOGY
(2022)
Article
Multidisciplinary Sciences
J. Boutin, J. Rosier, D. Cappellen, F. Prat, J. Toutain, P. Pennamen, J. Bouron, C. Rooryck, J. P. Merlio, I. Lamrissi-Garcia, G. Cullot, S. Amintas, V. Guyonnet-Duperat, C. Ged, J. M. Blouin, E. Richard, S. Dabernat, F. Moreau-Gaudry, A. Bedel
Summary: The study reveals the genotoxicity caused by CRISPR-Cas9 leading to loss of heterozygosity changes, while also highlighting the potential safety concerns of this technology for gene therapy.
NATURE COMMUNICATIONS
(2021)
Article
Multidisciplinary Sciences
Bo Zhang, Diyin Luo, Yu Li, Vanja Perculija, Jing Chen, Jinying Lin, Yangmiao Ye, Songying Ouyang
Summary: The study presents crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage, providing insights into the mechanisms of target DNA duplex unwinding, R-loop formation, and DNA cleavage. It also suggests that Cas12i1 forms a 19-bp long DNA-RNA heteroduplex with the crRNA guide region.
NATURE COMMUNICATIONS
(2021)
Article
Biotechnology & Applied Microbiology
Phuc Leo H. Vo, Carlotta Ronda, Sanne E. Klompe, Ethan E. Chen, Christopher Acree, Harris H. Wang, Samuel H. Sternberg
Summary: The study introduces a new INTEGRATE system that enables highly accurate and marker-free DNA integration in bacteria at high efficiency. By combining CRISPR arrays and multiple enzymes for multiplexed insertions and deletions, the potential applications of INTEGRATE in complex bacterial communities were demonstrated.
NATURE BIOTECHNOLOGY
(2021)
Article
Multidisciplinary Sciences
Yanbo Wang, John Mallon, Haobo Wang, Digvijay Singh, Myung Hyun Jo, Boyang Hua, Scott Bailey, Taekjip Ha
Summary: The research discovered that the HNH domain of CRISPR-Cas9 exhibits high flexibility post-DNA cleavage, exposing a 3' flap after cleavage of the non-target strand, potentially interacting with the DNA repair machinery. These findings reveal the regulatory role of DNA cleavage activity on Cas9 conformation and suggest potential applications in biotechnology.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Multidisciplinary Sciences
Nilmani Singh, Adriana Reyes-Ordonez, Michael A. Compagnone, Jesus F. Moreno Castillo, Benjamin J. Leslie, Taekjip Ha, Jie Chen
Summary: This study explores the specific interactions of human PH domain-containing proteins with PIPs and reveals unexpected lipid-binding specificity among these proteins. By analyzing the structural requirements and functional relevance of PIP binding, as well as using a prediction algorithm, the researchers demonstrate widespread PIP binding among human PH domains.
NATURE COMMUNICATIONS
(2021)
Article
Biochemistry & Molecular Biology
Christopher W. Akey, Digvijay Singh, Christna Ouch, Ignacia Echeverria, Ilona Nudelman, Joseph M. Varberg, Zulin Yu, Fei Fang, Yi Shi, Junjie Wang, Daniel Salzberg, Kangkang Song, Chen Xu, James C. Gumbart, Sergey Suslov, Jay Unruh, Sue L. Jaspersen, Brian T. Chait, Andrej Sali, Javier Fernandez-Martinez, Steven J. Ludtke, Elizabeth Villa, Michael P. Rout
Summary: This study provides a structure of the isolated yeast NPC and reveals how flexible connectors tie together different structural and functional layers. It also suggests an evolutionary and mechanistic link between assembly and transport. Additionally, the study identifies three major NPC variants that may signify functional specializations at the nuclear periphery.
Review
Biochemistry & Molecular Biology
Taekjip Ha, Christian Kaiser, Sua Myong, Bin Wu, Jie Xiao
Summary: Due to their unique abilities to manipulate, label, and image individual molecules, single-molecule techniques provide unprecedented access to elementary biological processes.
Article
Biochemistry & Molecular Biology
Sanne E. Klompe, Nora Jaber, Leslie Y. Beh, Jason T. Mohabir, Aude Bernheim, Samuel H. Sternberg
Summary: This study establishes a comprehensive bioinformatic and experimental pipeline to explore the diversity of Type I-F CRISPR-associated transposons. The study reports DNA integration for 20 systems and identifies a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. Additionally, the study reveals the presence of anti-phage defense systems encoded by transposons, suggesting a role in transmitting innate immunity between bacteria.
Article
Multidisciplinary Sciences
Li Dai, Digvijay Singh, Suoang Lu, Vishal Kottadiel, Reza Vafabakhsh, Marthandan Mahalingam, Yann R. Chemla, Taekjip Ha, Venigalla B. Rao
Summary: The study revealed that the active bacteriophage T4 DNA packaging motor is a pentamer and is able to tolerate inactive subunits. However, motors with inactive subunits showed decreased DNA engagements, higher failure rates in encapsidation, reduced packaging velocity, and increased pausing. This suggests that strict coordination among motor subunits is not crucial for the function of packaging motors.
NATURE COMMUNICATIONS
(2021)
Article
Chemistry, Multidisciplinary
Hossein Moghimianavval, Chintan Patel, Sonisilpa Mohapatra, Sung-Won Hwang, Tunc Kayikcioglu, Yashar Bashirzadeh, Allen P. Liu, Taekjip Ha
Summary: InterSpy is introduced as a synthetic biology tool for engineering membrane-membrane interfaces. It allows tracking and reconstitution of functional fluorescent protein between apposing synthetic or cell membranes, demonstrating the potential for designing non-native cellular communication pathways and creating synthetic tissues. The technology is tested using a mammalian cell-free expression system and shows promising results.
Article
Biology
Claudia C. Carcamo, Matthew F. Poyton, Anand Ranjan, Giho Park, Robert K. Louder, Xinyu A. Feng, Jee Min Kim, Thuc Dzu, Carl Wu, Taekjip Ha
Summary: This study characterizes the 1D scanning properties of SWR1, a chromatin remodeler, and reveals the impacts of various factors on its scanning process. By directly observing SWR1 diffusion on DNA, the study demonstrates that ATP and a DNA-binding subunit play important roles in the overall diffusive behavior of the complex. The findings provide valuable insights into the role of SWR1 in chromatin remodeling.
Article
Cell Biology
Roger S. Zou, Alberto Marin-Gonzalez, Yang Liu, Hans B. Liu, Leo Shen, Rachel K. Dveirin, Jay X. J. Luo, Reza Kalhor, Taekjip Ha
Summary: Here, we propose an approach that combines CRISPR system with high-throughput sequencing to target hundreds of epigenetically diverse endogenous genomic sites simultaneously, measuring Cas9 dynamics and cellular responses. The use of multi-target guide RNAs (mgRNAs) enables massive multiplexing of CRISPR, providing insights into Cas9 binding, cleavage, and cellular response to DNA damage.
NATURE CELL BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Momcilo Gavrilov, Joshua Y. C. Yang, Roger S. Zou, Wen Ma, Chun-Ying Lee, Sonisilpa Mohapatra, Jimin Kang, Ting-Wei Liao, Sua Myong, Taekjip Ha
Summary: Polymerase Chain Reaction (PCR) is a crucial method in molecular diagnostics and life sciences. This study introduces a new method, SHARP (SSB-Helicase Assisted Rapid PCR), which uses an engineered helicase and single-stranded DNA binding protein (SSB) to replace the heating step of PCR. The new method allows the isothermal amplification of DNA fragments up to 6000 base pairs.
NATURE COMMUNICATIONS
(2022)
Article
Chemistry, Physical
Ikenna C. Okafor, Taekjip Ha
Summary: CRISPR Cas9 is an RNA-guided endonuclease that is a part of bacterial adaptive immune system. By developing a single molecule FRET assay, the study investigated the conformational changes of sgRNA and the binding of Cas9 to sgRNA, providing insights into the assembly dynamics of Cas9 RNA ribonucleoprotein complex. This research could contribute to the rational design of sgRNAs and improve the editing outcomes.
JOURNAL OF PHYSICAL CHEMISTRY B
(2023)
Article
Biochemistry & Molecular Biology
Aakash Basu, Dmitriy G. Bobrovnikov, Basilio Cieza, Juan Pablo Arcon, Zan Qureshi, Modesto Orozco, Taekjip Ha
Summary: In this study, we comprehensively characterized the mechanical code of DNA using high-throughput experimental methods and developed a physical model to describe the sequence and methylation dependence of DNA deformation. Our measurements and model validations demonstrated that sequence and epigenetic modifications can encode regulatory information in diverse contexts.
NATURE STRUCTURAL & MOLECULAR BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Yanbo Wang, W. Taylor Cottle, Haobo Wang, Momcilo Gavrilov, Roger S. Zou, Minh-Tam Pham, Srinivasan Yegnasubramanian, Scott Bailey, Taekjip Ha
Summary: We describe a method called sgGOLDFISH for direct visualization of point mutations in situ, which can differentiate wild-type and mutant sequences with single-nucleotide sensitivity. This method has potential applications in studying genetic diseases and nuclear biology.
NATURE COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Aman Y. Husbands, Antje Feller, Vasudha Aggarwal, Courtney E. Dresden, Ashton S. Holub, Taekjip Ha, Marja C. P. Timmermans
Summary: The START domain of the HD-ZIPIII transcription factors plays a crucial role in promoting homodimerization and increasing transcriptional potency, while also binding to phospholipids. This discovery resolves a long-standing mystery in plant development and highlights the regulatory potential of this evolutionary module.