Article
Biotechnology & Applied Microbiology
Hui Kwon Kim, Goosang Yu, Jinman Park, Seonwoo Min, Sungtae Lee, Sungroh Yoon, Hyongbum Henry Kim
Summary: This study identified factors affecting PE2 efficiency through high-throughput evaluation and developed three computational models to predict pegRNA efficiency, which can be applied to edits of various types and positions. Spearman's correlations between 0.47 and 0.81 were found when testing the accuracy of the predictions using independent test data sets.
NATURE BIOTECHNOLOGY
(2021)
Article
Multidisciplinary Sciences
Zsolt Bodai, Alena L. Bishop, Valentino M. Gantz, Alexis C. Komor
Summary: Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. This study introduces a general strategy called the double tap method, which improves HDR-mediated precision editing efficiency by taking advantage of the reproducible nature of indel sequences. The method utilizes multiple gRNAs, including a primary gRNA that targets the wild-type genomic sequence and one or more secondary gRNAs that target the most common indel sequence(s).
NATURE COMMUNICATIONS
(2022)
Article
Engineering, Biomedical
Shuojun Li, Moqing Du, Jiamin Deng, Guiyun Deng, Jiaying Li, Zhiyong Song, Heyou Han
Summary: The CRISPR/Cas9 gene editing technology provides a promising strategy for Duchenne muscular dystrophy (DMD) therapy. This study introduces biomineralized nanoparticles (Bm-SpRY NPs) as a method for efficient DMD gene editing in vitro and in vivo, with advantages of biocompatibility and high delivery efficiency.
ACTA BIOMATERIALIA
(2022)
Article
Biotechnology & Applied Microbiology
Nahye Kim, Sungchul Choi, Sungjae Kim, Myungjae Song, Jung Hwa Seo, Seonwoo Min, Jinman Park, Sung-Rae Cho, Hyongbum Henry Kim
Summary: A deep learning model is used to predict the best base editor for specific applications. Seven base editors and nine Cas9 variants were systematically compared to determine their editing windows, outcomes, and preferred motifs. Two computational models, DeepCas9variants and DeepBE, were developed to predict the efficiency and outcomes of base editors, resulting in significantly higher efficiency for DeepBE-designed editors compared to rational design.
NATURE BIOTECHNOLOGY
(2023)
Article
Multidisciplinary Sciences
Zifeng Cui, Rui Tian, Zhaoyue Huang, Zhuang Jin, Lifang Li, Jiashuo Liu, Zheying Huang, Hongxian Xie, Dan Liu, Haiyan Mo, Rong Zhou, Bin Lang, Bo Meng, Haiyan Weng, Zheng Hu
Summary: Genome editing technologies have great potential in biomedical research and drug development. This study introduces a CRISPR/Cas9 tool called FrCas9 from Faecalibaculum rodentium, which has efficient and specific gene editing capabilities and can be used for the treatment of TATA-box related diseases.
NATURE COMMUNICATIONS
(2022)
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
Biochemistry & Molecular Biology
Qian Wang, Jin Liu, Josephine M. Janssen, Marie Le Bouteiller, Richard L. Frock, Manuel A. F. Goncalves
Summary: The study demonstrates that high-specificity Cas9 nucleases can be converted into nicking Cas9(D10A) variants with superior precision compared to the commonly used Cas9(D10A)nickase. Dual nicking RGNs based on these Cas9(D10A) variants can achieve gene knockouts and knock-ins at frequencies similar to or higher than conventional methods. Additionally, the high-specificity dual nicking RGNs are able to distinguish highly similar sequences and preserve genomic integrity.
NUCLEIC ACIDS RESEARCH
(2021)
Article
Biotechnology & Applied Microbiology
Siqi Gao, Yao Wang, Tao Qi, Jingjing Wei, Ziying Hu, Jingtong Liu, Shuna Sun, Huihui Liu, Yongming Wang
Summary: In this study, the PAM sequences of CjCas9 and its orthologs, Hsp1Cas9, Hsp2Cas9, and CcuCas9, were investigated. It was found that they recognized N4RAA, N4CNA, and N4CY PAMs, respectively. By analyzing the crystal structure of CjCas9, eight mutations were identified to improve specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.
Article
Immunology
Huan Qin, Wenliang Zhang, Shiyao Zhang, Yuan Feng, Weihui Xu, Jia Qi, Qian Zhang, Chunxiu Xu, Shanshan Liu, Jia Zhang, Yushuang Lei, Wanqin Liu, Shuyu Feng, Jingjing Wang, Xuefei Fu, Zifen Xu, Ping Li, Kai Yao
Summary: The researchers developed a genome-editing tool called PESpRY, which combines the versatility of prime editors with the unconstrained PAM requirement of the SpRY Cas9 variant. Using this tool, they were able to successfully correct gene mutations in a mouse model of retinitis pigmentosa, leading to substantial restoration of vision.
JOURNAL OF EXPERIMENTAL MEDICINE
(2023)
Review
Biochemistry & Molecular Biology
Kirti Prasad, Anila George, Nithin Sam Ravi, Kumarasamypet M. Mohankumar
Summary: CRISPR/Cas9 system, developed from bacterial adaptive immune system, has revolutionized genome engineering and made gene therapy closer to reality. Its rapid evolution from double strand breaks to advanced gene regulation applications has greatly benefited medical science.
MOLECULAR BIOLOGY REPORTS
(2021)
Review
Biotechnology & Applied Microbiology
Rongming Liu, Liya Liang, Emily F. Freed, Ryan T. Gill
Summary: CRISPR technology, a universal tool for genome engineering, has been revolutionizing biotechnology. The diverse Cas proteins with complex structures contain multiple evolutionarily conserved functional domains, and engineering key domains can modify the functions of CRISPR/Cas systems.
TRENDS IN BIOTECHNOLOGY
(2021)
Review
Medicine, Research & Experimental
Kiran Musunuru
Summary: The rapid development of genome-editing technologies has opened up new possibilities for treating cardiovascular diseases. This review discusses various genome-editing approaches and their potential applications in cardiovascular disease treatment, providing insights into the path from technology development to clinical trials.
JOURNAL OF CLINICAL INVESTIGATION
(2022)
Article
Biochemistry & Molecular Biology
Cedric Happi Mbakam, Joel Rousseau, Guillaume Tremblay, Pouire Yameogo, Jacques P. Tremblay
Summary: Prime editing, derived from the CRISPR/Cas9 discovery, allows for the modification of selected nucleotides in a specific gene. It can be used to correct point mutations in the DMD gene.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Plant Sciences
Johannes Stuttmann, Karen Barthel, Patrick Martin, Jana Ordon, Jessica L. Erickson, Rosalie Herr, Filiz Ferik, Carola Kretschmer, Thomas Berner, Jens Keilwagen, Sylvestre Marillonnet, Ulla Bonas
Summary: Genome editing using RNA-guided nucleases like SpCas9 has been successful in various plant species. A toolkit based on an optimized zCas9i gene allows targeting multiple independent loci simultaneously. Efficient multiplexing was achieved in Nicotiana benthamiana and Arabidopsis thaliana, with the latter showing lower efficiency due to Cas9 availability limitations. This study opens new possibilities for generating complex genotypes through multiplexing.
Article
Plant Sciences
Guo-Qing Song, Grace Urban, John T. Ryner, Gan-Yuan Zhong
Summary: This study evaluated chimeric editing in gene editing, using a transgenic tobacco line and a CRISPR-Cas9 editing vector. It was found that chimeric editing primarily occurred during or after cell regeneration. Further transformation was conducted to enhance editing efficiency. The findings provide valuable information for understanding gene editing in plant cells using constitutively expressed CRISPR-Cas9 and gRNAs.
Article
Biochemistry & Molecular Biology
Ignazio Maggio, Hidde A. Zittersteijn, Qian Wang, Jin Liu, Josephine M. Janssen, Ivan Toral Ojeda, Silvere M. van der Maarel, Arjan C. Lankester, Rob C. Hoeben, Manuel A. F. V. Goncalves
Article
Biochemistry & Molecular Biology
Xiaoyu Chen, Francesca Tasca, Qian Wang, Jin Liu, Josephine M. Janssen, Marcella D. Brescia, Milena Bellin, Karoly Szuhai, Josefin Kenrick, Richard L. Frock, Manuel A. F. Goncalves
NUCLEIC ACIDS RESEARCH
(2020)
Review
Cell Biology
Francesca Tasca, Qian Wang, Manuel A. F. V. Goncalves
Review
Endocrinology & Metabolism
Hidde A. Zittersteijn, Manuel A. F. V. Goncalves, Rob C. Hoeben
Summary: Genetic therapies based on gene addition have achieved clinical successes, especially using viral vector-based gene delivery systems. Emerging gene editing approaches like CRISPR-Cas9 nucleases and new nickases are also gaining traction in the field.
JOURNAL OF INHERITED METABOLIC DISEASE
(2021)
Article
Biochemistry & Molecular Biology
Marcella Brescia, Ying-Chi Chao, Andreas Koschinski, Jakub Tomek, Manuela Zaccolo
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2020)
Article
Biochemistry & Molecular Biology
Qian Wang, Jin Liu, Josephine M. Janssen, Marie Le Bouteiller, Richard L. Frock, Manuel A. F. Goncalves
Summary: The study demonstrates that high-specificity Cas9 nucleases can be converted into nicking Cas9(D10A) variants with superior precision compared to the commonly used Cas9(D10A)nickase. Dual nicking RGNs based on these Cas9(D10A) variants can achieve gene knockouts and knock-ins at frequencies similar to or higher than conventional methods. Additionally, the high-specificity dual nicking RGNs are able to distinguish highly similar sequences and preserve genomic integrity.
NUCLEIC ACIDS RESEARCH
(2021)
Article
Cell Biology
Jin Ma, Gerard van der Zon, Manuel A. F. V. Goncalves, Maarten van Dinther, Midory Thorikay, Gonzalo Sanchez-Duffhues, Peter ten Dijke
Summary: This study demonstrates that TGF-beta 2 is crucial for triggering EndMT by upregulating SNAIL, while SLUG plays a lesser role. Additionally, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. This suggests that a balance between SNAIL/SLUG and ID proteins determines the control over endothelial vs. mesenchymal cell states.
FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Qian Wang, Jin Liu, Josephine M. Janssen, Francesca Tasca, Hailiang Mei, Manuel A. F. Goncalves
Summary: Packaging prime editing constructs in adenoviral capsids overcomes the difficulty in delivering large prime editing complexes into cells, resulting in robust genome editing with high efficiencies in human cells. The correlation between prime editing activity and cellular replication was found, and the proportion between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context.
NUCLEIC ACIDS RESEARCH
(2021)
Article
Biochemistry & Molecular Biology
Francesca Tasca, Marcella Brescia, Qian Wang, Jin Liu, Josephine M. Janssen, Karoly Szuhai, Manuel A. F. Goncalves
Summary: This study investigates the use of viral gene-deleted adenovector particles (AdVPs) as sources of CRISPR-Cas9 nucleases and donor DNA constructs for targeted chromosomal insertion in human cells. The researchers found that donors prone to homology-mediated end joining (HMEJ) yielded higher genome editing frequencies in muscle progenitor cells, while in induced pluripotent stem cells (iPSCs), both homologous recombination (HR) and HMEJ substrates showed similar editing levels. Furthermore, p53 inhibition increased HMEJ-based genome editing in iPSCs. Overall, AdVPs provide a robust platform for large genomic edits in human cells.
NUCLEIC ACIDS RESEARCH
(2022)
Article
Biochemistry & Molecular Biology
Qian Wang, Jin Liu, Josephine M. Janssen, Manuel A. F. Goncalves
Summary: Homology-directed recombination (HDR) allows for precise genomic edits in mammalian cells, but programmable nucleases can also cause unintended modifications. In trans paired nicking (ITPN) using CRISPR-Cas9 nickases enables seamless genome editing. This study identified high-specificity CRISPR-Cas9 nucleases for precise genome editing and demonstrated the compatibility of ITPN with these nickases, leading to editing of essential and recurrent sequences without activating negative DNA damage responses in human iPSCs.
NUCLEIC ACIDS RESEARCH
(2023)
Editorial Material
Medicine, Research & Experimental
Zhen Li, Manuel A. F. V. Goncalves
MOLECULAR THERAPY-NUCLEIC ACIDS
(2023)
Article
Medicine, Research & Experimental
Francesca Tasca, Marcella Brescia, Jin Liu, Josephine M. Janssen, Kamel Mamchaoui, Manuel A. F. V. Goncalves
Summary: Genome editing based on dual CRISPR-Cas9 complexes allows for the removal of specific genomic sequences in living cells, but delivering the required reagents in a synchronous and stoichiometric manner remains challenging. The delivery of forced Cas9 heterodimers or forced CRISPR-Cas9 heterodimers using high-capacity adenovector particles (AdVPs) promotes precise multiplexing genome editing and achieves superior efficiency and accuracy compared to separate delivery of components. The integrated delivery of forced CRISPR-Cas9 heterodimers engages target sequences in a more coordinated fashion, resulting in successful gene editing outcomes.
MOLECULAR THERAPY-NUCLEIC ACIDS
(2023)
Article
Biochemical Research Methods
Arend W. Overeem, Yolanda W. Chang, Ioannis Moustakas, Celine M. Roelse, Sanne Hillenius, Talia Van Der Helm, Valerie F. Van Der Schrier, Manuel A. F. V. Goncalves, Hailiang Mei, Christian Freund, Susana M. Chuva de Sousa Lopes
Summary: Current methods for generating human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) are inefficient, making it challenging to produce enough hPGCLCs for in vitro gametogenesis optimization. In this study, a differentiation method using diluted basement membrane extract (BMEx) and low BMP4 concentration was developed, which efficiently induced hPGCLC differentiation in scalable 2D cell culture. The overlay of BMEx enhanced BMP/SMAD signaling, induced lumenogenesis, and increased the expression of key hPGCLC-progenitor markers. These findings highlight the significance of BMEx in hPGCLC differentiation and its potential for studying PGC and amnion formation, as well as investigating further steps towards achieving in vitro gametogenesis.
CELL REPORTS METHODS
(2023)