Article
Biotechnology & Applied Microbiology
Belen Esquerra-Ruvira, Ignacio Baquedano, Raul Ruiz, Almudena Fernandez, Lluis Montoliu, Francisco J. M. Mojica
Summary: Non-coding RNAs (crRNAs) produced from CRISPR loci and Cas proteins interfere with the spread of genetic elements. The programmable targeting of nucleic acids by ribonucleoproteins enables the modification of DNA and RNA targets. The identification of a smaller Cas9 protein (EHCas9) associated with a new CRISPR-Cas9 system offers a simplified tool for genome editing in prokaryotes and eukaryotic cells.
MICROBIAL BIOTECHNOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Keiji Nishida, Akihiko Kondo
Summary: CRISPR-Cas system offers various modes for DNA editing in metabolic engineering, with consideration of host-dependent repair pathways. The versatility of CRISPR system has led to derivative technologies to complement nuclease-based editing, avoiding cytotoxicity, especially in microorganisms.
METABOLIC ENGINEERING
(2021)
Article
Biotechnology & Applied Microbiology
Youxiang Liang, Yuwen Wei, Song Jiao, Huimin Yu
Summary: The failure of conventional CRISPR/Cas9 technologies in Rhodococcus opacus was mainly due to the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks. An efficient single-stranded DNA recombineering system coupled with CRISPR/Cas9 counter-selection was successfully developed for rapid and scarless editing of the R. opacus genome. This novel tool enabled gene disruption, base mutation, and simultaneous disruption of double genes, with editing efficiencies ranging from 22% to 100%.
SYNTHETIC AND SYSTEMS BIOTECHNOLOGY
(2021)
Article
Microbiology
Xueli Zhang, Chaohui Zhang, Caijiao Liang, Bizhou Li, Fanmei Meng, Yuncan Ai
Summary: Bacteriophages, the most abundant organisms in the biosphere, have been sequenced extensively. However, the study of bacteriophage functional genomics has been hindered by a lack of effective research methods. This study designed a phage genome editing platform based on the CRISPR-Cas9 system, and successfully achieved gene editing in Vibrio natriegens phage TT4P2. This platform has the potential to advance research on phage gene diversity and accelerate the development of phage synthetic biology and nanotechnology.
MICROBIOLOGY SPECTRUM
(2022)
Letter
Biochemical Research Methods
Qi Li, Mingjun Sun, Lu Lv, Yong Zuo, Suyi Zhang, Ying Zhang, Sheng Yang
Summary: Due to its high specificity and programmability, CRISPR-Cas-based antimicrobials have been developed to eliminate specific strains, such as antibiotic-resistant bacteria, in the microbiome. However, the generation of escapers has resulted in lower elimination efficiency than the recommended acceptable rate. In this study, the escaping mechanisms in Escherichia coli were investigated, and strategies for reducing the escapers were devised accordingly.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Hyun-Woo Je, Chang-Hun Ji, Jun-Yong Kim, Hahk-Soo Kang
Summary: The CRISPR/Cas9 system faces toxicity issues in Streptomyces genome engineering due to overexpression of the Cas9 protein. The CaExTun platform has been developed to optimize Cas9 expression for reduced toxicity while maintaining sufficient DSB activity. This tool enables rapid optimization of the CRISPR/Cas9 system in a species-specific manner, facilitating genome engineering efforts in Streptomyces.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Anastasiya Matveeva, Alexander Ryabchenko, Viktoria Petrova, Daria Prokhorova, Evgenii Zhuravlev, Alexander Zakabunin, Artem Tikunov, Grigory Stepanov
Summary: Research on Cas9 nucleases from different organisms has the potential to advance genome engineering and gene therapy tools. Thermophilic Cas9 nucleases, which have varying activity temperature ranges and PAM preferences, are expanding. This study focuses on constructing a compact Cas9 nuclease that displays nuclease activity in the 37-60 degrees C range.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Review
Cell Biology
Gemma Llargues-Sistac, Laia Bonjoch, Sergi Castellvi-Bel
Summary: The use of next-generation sequencing (NGS) technologies has greatly advanced our understanding of the mutational landscape of complex human diseases such as cancer. Haploid human cell models, such as the HAP1 cell line, have emerged as valuable tools for functional gene studies, especially in combination with CRISPR-Cas9 gene editing technology. This review explores the recent applications of the HAP1 cell line in functional genetic studies and high-throughput genetic screens, highlighting its potential to enhance our understanding of gene function and the genetic basis of human diseases identified through NGS technologies, and its implications for clinical practice and patient care.
FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Hyo-Ju Jang, Hye-Jeong Park, Hong Seok Choi, Hyun Jun Jung, Tae-Hwan Kwon
Summary: This study aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology. The results showed that these cells had higher abundance of AQP2 protein in the absence of stimulation and exhibited normal response to dDAVP stimulation.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Biotechnology & Applied Microbiology
Guoyi Zhang, Junyu Wang, Yiwen Li, Guangdong Shang
Summary: Site-directed and saturation mutagenesis are important DNA methodologies for studying protein structure and function. Existing plasmid-based gene mutation methods involve tedious cloning steps, while oligo-mediated recombineering provides a more efficient and simplified approach. CRISPR/Cas9-assisted ssDNA recombineering offers a high-efficiency and versatile strategy for site-directed and saturation mutagenesis.
BIOTECHNOLOGY LETTERS
(2023)
Review
Chemistry, Physical
Noshad Peyravian, Maziar Malekzadeh Kebria, Jafar Kiani, Peiman Brouki Milan, Masoud Mozafari
Summary: This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations, highlighting the limitations of conventional delivery tools and the advantages of microfluidic systems in in vitro delivery.
Article
Biochemistry & Molecular Biology
Azamat V. Karginov, Marina G. Tarutina, Anastasia R. Lapteva, Maria D. Pakhomova, Artur A. Galliamov, Sergey Y. Filkin, Alexey N. Fedorov, Michael O. Agaphonov
Summary: In this work, an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts is presented. The vector set includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of sgRNA gene for a specific target sequence only requires the insertion of a 24 bp oligonucleotide duplex into the scaffold. The functionality of this vector set has been demonstrated in multiple methylotrophic yeast species.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Biotechnology & Applied Microbiology
Junchang Su, Pengju Wang, Ju Li, Dongdong Zhao, Siwei Li, Feiyu Fan, Zhubo Dai, Xiaoping Liao, Zhitao Mao, Chunzhi Zhang, Changhao Bi, Xueli Zhang
Summary: In this study, a simple CRISPR-based genome engineering technique was developed to construct multi-chromosomal E. coli strains without heterologous genetic parts. The successful creation of a new E. coli strain using this technique has important implications for synthetic biology research and applications.
MICROBIAL CELL FACTORIES
(2022)
Article
Biochemistry & Molecular Biology
Patrick Ferreira, Altino Branco Choupina
Summary: In recent years, the number of tools and techniques for precise genome editing has increased significantly. The CRISPR/Cas9 technology has gained much attention due to its simplicity, affordability, and effectiveness. It has the potential for various applications in gene editing, including functional genomics, cell model development, targeted therapies, and improvement of agricultural and environmental species.
MOLECULAR BIOLOGY REPORTS
(2022)
Article
Chemistry, Multidisciplinary
Ruosen Xie, Xiuxiu Wang, Yuyuan Wang, Mingzhou Ye, Yi Zhao, Brian S. Yandell, Shaoqin Gong
Summary: This study reports the development of a facilely fabricated pH-responsive polymer nanoparticle for safe and efficient delivery of CRISPR-associated protein 9 (Cas9) for gene editing. These nanoparticles possess many desirable properties, making them potential candidates for clinical applications in genome editing.
ADVANCED MATERIALS
(2022)
Article
Biochemical Research Methods
Tomas Aparicio, Victor de Lorenzo, Esteban Martinez-Garcia
BIOTECHNOLOGY JOURNAL
(2019)
Review
Biochemical Research Methods
Esteban Martinez-Garcia, Victor de Lorenzo
CURRENT OPINION IN BIOTECHNOLOGY
(2019)
Article
Microbiology
Tomas Aparicio, Akos Nyerges, Istvan Nagy, Csaba Pal, Esteban Martinez-Garcia, Victor de Lorenzo
ENVIRONMENTAL MICROBIOLOGY
(2020)
Article
Biochemistry & Molecular Biology
Esteban Martinez-Garcia, Angel Goni-Moreno, Bryan Bartley, James McLaughlin, Lucas Sanchez-Sampedro, Hector Pascual del Pozo, Clara Prieto Hernandez, Ada Serena Marletta, Davide De Lucrezia, Guzman Sanchez-Fernandez, Sofia Fraile, Victor de Lorenzo
NUCLEIC ACIDS RESEARCH
(2020)
Article
Immunology
Stephanie Crone, Martha Vives-Florez, Lasse Kvich, Aaron M. Saunders, Matthew Malone, Mette H. Nicolaisen, Esteban Martinez-Garcia, Catalina Rojas-Acosta, Maria Catalina Gomez-Puerto, Henrik Calum, Marvin Whiteley, Roberto Kolter, Thomas Bjarnsholt
Correction
Biochemistry & Molecular Biology
Esteban Martinez-Garcia, Angel Goni-Moreno, Bryan Bartley, James McLaughlin, Lucas Sanchez-Sampedro, Hector Pascual del Pozo, Clara Prieto Hernandez, Ada Serena Marletta, Davide De Lucrezia, Guzman Sanchez-Fernandez, Sofia Fraile, Victor de Lorenzo
NUCLEIC ACIDS RESEARCH
(2020)
Article
Microbiology
See-Yeun Ting, Esteban Martinez-Garcia, Shuo Huang, Savannah K. Bertolli, Katherine A. Kelly, Kevin J. Cutler, Elizabeth D. Su, Hui Zhi, Qing Tang, Matthew C. Radey, Manuela Raffatellu, S. Brook Peterson, Victor de Lorenzo, Joseph D. Mougous
CELL HOST & MICROBE
(2020)
Article
Microbiology
Coral Garcia-Gutierrez, Tomas Aparicio, Lucia Torres-Sanchez, Esteban Martinez-Garcia, Victor de Lorenzo, Claudio J. Villar, Felipe Lombo
Article
Biochemical Research Methods
David R. Espeso, Elena Algar, Esteban Martinez-Garcia, Victor de Lorenzo
BMC BIOINFORMATICS
(2020)
Article
Biochemistry & Molecular Biology
Elena Algar, Yamal Al-Ramahi, Victor de Lorenzo, Esteban Martinez-Garcia
Article
Biochemical Research Methods
Esteban Martinez-Garcia, Sofia Fraile, David Rodriguez Espeso, Davide Vecchietti, Giovanni Bertoni, Victor de Lorenzo
ACS SYNTHETIC BIOLOGY
(2020)
Article
Biochemical Research Methods
Sofia Fraile, Maria Briones, Monica Revenga-Parra, Victor de Lorenzo, Encarnacion Lorenzo, Esteban Martinez-Garcia
Summary: Gram-negative bacteria have complex outer membrane structures that make designing reliable bacterial coatings challenging. In this study, ectopic expression of a fibrinogen-specific nanobody on Pseudomonas putida cells enabled the formation of a bacterial monolayer strongly bound to an antigen-coated support. This approach has potential for engineering specific bacterial coverings on target surfaces.
ACS SYNTHETIC BIOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Esteban Martinez-Garcia, Sofia Fraile, Elena Algar, Tomas Aparicio, Elena Velazquez, Belen Calles, Huseyin Tas, Blas Blazquez, Bruno Martin, Clara Prieto, Lucas Sanchez-Sampedro, Morten H. H. Norholm, Daniel C. Volke, Nicolas T. Wirth, Pavel Dvorak, Lorea Alejaldre, Lewis Grozinger, Matthew Crowther, Angel Goni-Moreno, Pablo Nikel, Juan Nogales, Victor de Lorenzo
Summary: The SEVA platform, launched a decade ago, is a database and physical repository for genetic analysis and engineering. The latest update includes more plasmids and expanded features, making it a popular resource for genetic analysis, genome editing, and bioengineering research.
NUCLEIC ACIDS RESEARCH
(2023)
Article
Multidisciplinary Sciences
Tomas Aparicio, Akos Nyerges, Esteban Martinez-Garcia, Victor de Lorenzo
Article
Biology
David R. Espeso, Esteban Martinez-Garcia, Victor de Lorenzo