Article
Biotechnology & Applied Microbiology
Yuan Zong, Yijing Liu, Chenxiao Xue, Boshu Li, Xiangyang Li, Yanpeng Wang, Ji Li, Guanwen Liu, Xingxu Huang, Xiaofeng Cao, Caixia Gao
Summary: In this study, we have improved the editing efficiency of prime editing by engineering the structure of the prime editor and combining it with specific guide RNAs. The optimized prime editor showed significantly increased editing efficiency in plant cells, and when combined with engineered guide RNAs, it resulted in the generation of herbicide-tolerant rice plants.
NATURE BIOTECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Shengyao Zhi, Yuxi Chen, Guanglan Wu, Jinkun Wen, Jinni Wu, Qianyi Liu, Yang Li, Rui Kang, Sihui Hu, Jiahui Wang, Puping Liang, Junjiu Huang
Summary: Prime editor (PE) is a new genome editing tool that has the potential to correct the majority of known human genetic disease-related mutations. In this study, split-PEs were constructed and delivered using dual adenoassociated viruses (AAVs), successfully mediating gene editing in human cells and adult mouse retina.
Article
Biotechnology & Applied Microbiology
Chunwei Zheng, Shun-Qing Liang, Bin Liu, Pengpeng Liu, Suet-Yan Kwan, Scot A. Wolfe, Wen Xue
Summary: Prime editor (PE) has great potential for gene therapy, but it is challenging to deliver PE in vivo. In this study, a compact PE without the RNase H domain was developed, which showed comparable editing efficiency with full-length PE. Using a Cas9 split site, robust editing was achieved in both cells and mouse liver. Furthermore, the compact PE efficiently mediated gene insertion in mouse liver without the stop codon read-through effect observed with full-length PE, indicating its potential for advancing prime editing in vivo.
Article
Cell Biology
Desiree Boeck, Tanja Rothgangl, Lukas Villiger, Lukas Schmidheini, Mai Matsushita, Nicolas Mathis, Eleonora Ioannidi, Nicole Rimann, Hiu Man Grisch-Chan, Susanne Kreutzer, Zacharias Kontarakis, Manfred Kopf, Beat Thoeny, Gerald Schwank
Summary: Prime editing is a highly versatile CRISPR-based technology that is able to edit genomes without causing DNA double-strand breaks. In this study, the researchers developed a smaller version of the SpCas9 prime editor and used a viral delivery system to edit genes in the liver. They were able to successfully correct a disease-causing mutation in a mouse model of phenylketonuria, leading to therapeutic reduction of phenylalanine in the blood. Although there are limitations to the current approach, further development of the technology may offer potential treatments for genetic liver diseases.
SCIENCE TRANSLATIONAL MEDICINE
(2022)
Article
Biotechnology & Applied Microbiology
Pei Ni, Yidi Zhao, Ximeng Zhou, Zehua Liu, Zhengwei Huang, Zhongfu Ni, Qixin Sun, Yuan Zong
Summary: Researchers developed an upgraded engineered plant prime editor, ePPEplus, by introducing a V223A substitution into reverse transcriptase in hexaploid wheat, which enhances editing efficiency by an average of 33.0-fold compared to the original PPE and 6.4-fold compared to ePPE. Importantly, they established a robust multiplex prime editing platform for simultaneous editing of multiple genes, achieving editing frequencies up to 74.5% in regenerated wheat plants, thus expanding the applicability of prime editors for stacking agronomic traits.
Article
Biotechnology & Applied Microbiology
Bryan P. Simpson, Carolyn M. Yrigollen, Aleksandar Izda, Beverly L. Davidson
Summary: Using polymerase-free, targeted long-read nanopore sequencing, we evaluated the editing effects of single and dual gRNA AAV-CRISPR on the human ATXN2 gene in transgenic mouse models of SCA2. The results showed an editing rate of 10%-25% and identified AAV integration, large deletions, and transgene rearrangements as unexpected outcomes. PCR-based nanopore sequencing, in contrast, showed a bias for partial AAV fragments and inverted terminal repeats and failed to detect full-length AAV. This study provides a comprehensive understanding of the outcome spectrum of CRISPR editing in the mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.
Article
Multidisciplinary Sciences
F. Chemello, A. C. Chai, H. Li, C. Rodriguez-Caycedo, E. Sanchez-Ortiz, A. Atmanli, A. A. Mireault, N. Liu, R. Bassel-Duby, E. N. Olson
Summary: This study demonstrated the use of adenine base editor to restore dystrophin expression in cardiomyocytes affected by Duchenne muscular dystrophy mutation, as well as reframing the dystrophin open reading frame. In vivo experiments with mice showed successful gene editing and disease correction, indicating the potential of nucleotide editing for diverse DMD mutations with minimal genome modification.
Article
Biochemistry & Molecular Biology
Peter J. Chen, Jeffrey A. Hussmann, Jun Yan, Friederike Knipping, Purnima Ravisankar, Pin-Fang Chen, Cidi Chen, James W. Nelson, Gregory A. Newby, Mustafa Sahin, Mark J. Osborn, Jonathan S. Weissman, Britt Adamson, David R. Liu
Summary: The study found that DNA mismatch repair (MMR) hinders prime editing and promotes undesired byproducts, but the efficiency of substitution, small insertion, and small deletion edits can be enhanced by developing PE4 and PE5 prime editing systems. Strategic silent mutations near the intended edit and optimization of prime editor protein also contribute to improved editing outcomes.
Article
Multidisciplinary Sciences
Mu Li, Aaron Zhong, Youjun Wu, Mega Sidharta, Michael Beaury, Xiaolan Zhao, Lorenz Studer, Ting Zhou
Summary: Li et al. found that co-delivering a dominant negative fragment of p53 (p53DD) greatly enhances the efficiency of precise editing in human pluripotent stem cells for prime editing and cytosine base editing, without compromising the genome-wide safety.
NATURE COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Karthikeyan Ponnienselvan, Pengpeng Liu, Thomas Nyalile, Sarah Oikemus, Stacy A. Maitland, Nathan D. Lawson, Jeremy Luban, Scot A. Wolfe
Summary: This study demonstrates that the length of the primer binding site (PBS) and the interaction between the PBS and the spacer sequence affects the efficiency of prime editing using ribonucleoprotein complexes. By reducing the complementarity between the PBS-spacer region, the auto-inhibitory interaction can be destabilized and prime editing efficiency is enhanced. In mammalian cells, a shorter PBS length with a PBS-target strand melting temperature near 37 degrees C is optimal for prime editing.
NUCLEIC ACIDS RESEARCH
(2023)
Article
Biochemistry & Molecular Biology
Kaiqin She, Yi Liu, Qinyu Zhao, Xiu Jin, Yiliu Yang, Jing Su, Ruiting Li, Li Song, Jianlu Xiao, Shaohua Yao, Fang Lu, Yuquan Wei, Yang Yang
Summary: In this study, the prime editor was optimized and successfully used to correct disease-causing mutations in inherited retinal diseases, restoring retinal and visual function. This has significant therapeutic potential.
SIGNAL TRANSDUCTION AND TARGETED THERAPY
(2023)
Article
Biochemistry & Molecular Biology
Jordan L. Doman, Smriti Pandey, Monica E. Neugebauer, Meirui An, Jessie R. Davis, Peyton B. Randolph, Amber Mcelroy, Xin D. Gao, Aditya Raguram, Michelle F. Richter, Kelcee A. Everette, Samagya Banskota, Kathryn Tian, Y. Allen Tao, Jakub Tolar, Mark J. Osborn, David R. Liu
Summary: Prime editing enables precise genome edits in living cells, and protein evolution and engineering have been used to generate prime editors with improved efficiency and reduced size. Different types of reverse transcriptases specialize in different types of edits, and the new editors show enhanced therapeutic editing. The improvements in prime editing have significant implications for gene editing technologies.
Article
Oncology
Alexandra G. Kouroukli, Nivethika Rajaram, Pavel Bashtrykov, Helene Kretzmer, Reiner Siebert, Albert Jeltsch, Susanne Bens
Summary: The study demonstrates the feasibility of silencing TERT alleles using allele-specific epigenome editing. This new strategy may have important advantages in cancer treatment, such as reducing adverse side effects.
CLINICAL EPIGENETICS
(2023)
Article
Biochemistry & Molecular Biology
Santiago C. Lopez, Kate D. Crawford, Sierra K. Lear, Santi Bhattarai-Kline, Seth L. Shipman
Summary: Modifications to the retron non-coding RNA result in increased production of reverse-transcribed DNA, leading to higher efficiency in genome editing in both prokaryotic and eukaryotic cells. The findings demonstrate the applicability of retron RT-DNA for precise editing of human cells and provide a general framework for using retrons in genome modification.
NATURE CHEMICAL BIOLOGY
(2022)
Article
Medicine, Research & Experimental
Qian Li, Jing Su, Yi Liu, Xiu Jin, Xiaomei Zhong, Li Mo, Qingnan Wang, Hongxin Deng, Yang Yang
Summary: This study engineered an all-in-one self-cleavage AAV-CRISPR-Cas9 system to restrict the expression of Cas9 nuclease, demonstrating a reduction in Cas9 protein expression and off-target activity while maintaining on-target editing efficacy in vivo. The inclusion of this self-cleavage system could enhance the safety profile of AAV-delivered genome-editing nucleases and facilitate their clinical translation.
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
(2021)
Article
Hematology
Anne Bruun Rovsing, Emil Aagaard Thomsen, Ian Nielsen, Thomas Wisbech Skov, Yonglun Luo, Karen Dybkaer, Jacob Giehm Mikkelsen
Summary: Using CRISPR screening, we uncover the cellular response to vincristine in DLBCL, identifying genes related to mitotic spindle organization and the mechanism behind vincristine resistance involving KIF18B and USP28. Our findings provide potential drug targets and mechanisms for future drug regimens.
BRITISH JOURNAL OF HAEMATOLOGY
(2023)
Article
Allergy
Laura Barrett Ryo, Didde Haslund, Anne Bruun Rovsing, Rasmus Pihl, Wariya Sanrattana, Steven de Maat, Yaseelan Palarasah, Coen Maas, Steffen Thiel, Jacob Giehm Mikkelsen
Summary: This study investigates the trans-inhibitory effects of different SERPING1 variants on the expression, secretion, functionality, and intracellular localization of C1 inhibitor (C1INH) in HeLa cells. The findings suggest that different SERPING1 variants may drive the pathogenicity of hereditary angioedema through different and sometimes overlapping molecular disease mechanisms, with some variants acting as dominant-negative factors.
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
(2023)
Article
Oncology
Eileen Wedge, Ulvi Ahmadov, Thomas B. Hansen, Zongliang Gao, Morten Tulstrup, Christophe Come, Sridhar Nonavinkere Srivatsan, Tanzir Ahmed, Jakob S. Jespersen, Balthasar C. Schlotmann, Claudia Schollkopf, Klas Raaschou-Jensen, Niels Odum, Jorgen Kjems, Rasmus O. Bak, Matthew J. Walter, Kirsten Gronbaek, Lasse S. Kristensen
Summary: Mutations in the U2AF1 gene are associated with a higher occurrence of myelodysplastic neoplasms (MDS) and a worse prognosis, but the exact molecular mechanisms are not fully understood. This study found that U2AF1 mutations may impact circRNA production, leading to increased cancer development. Increased circRNA expression levels were observed in cells and patient samples with U2AF1 mutations, suggesting a potential role of circRNA as a biomarker and therapeutic target in MDS.
Review
Biotechnology & Applied Microbiology
Louise Bendixen, Trine I. Jensen, Rasmus O. Bak
Summary: The CRISPR-Cas system has revolutionized gene editing and therapy with its ability to precisely cleave DNA. Newer versions of the technology, such as CRISPRa and CRISPRi, have enabled programmable transcriptional modulation and genetic screening. This review highlights the creation and delivery methods of these transcriptional modulators, recent technological advancements, and their applications in understanding genetic networks and diseases. The potential therapeutic uses of transcriptional modulation and future directions are also discussed.
Article
Multidisciplinary Sciences
Mingyu He, Kate Roussak, Feiyang Ma, Nicholas Borcherding, Vince Garin, Mike White, Charles Schutt, Trine I. Jensen, Yun Zhao, Courtney A. Iberg, Kairav Shah, Himanshi Bhatia, Daniel Korenfeld, Sabrina Dinkel, Judah Gray, Alina Ulezko Antonova, Stephen Ferris, David Donermeyer, Cecilia Lindestam Arlehamn, Matthew M. Gubin, Jingqin Luo, Laurent Gorvel, Matteo Pellegrini, Alessandro Sette, Thomas Tung, Rasmus Bak, Robert L. Modlin, Ryan C. Fields, Robert D. Schreiber, Paul M. Allen, Eynav Klechevsky
Summary: The reduction of human CD1c+CD5+ dendritic cells (DCs) in melanoma-affected lymph nodes and the correlation between CD5 expression on DCs and patient survival have been observed. Activating CD5 on DCs enhances T cell priming and improves survival after immune checkpoint blockade (ICB) therapy. CD5+ DCs are essential for optimal ICB therapy.
Article
Biology
Francisco X. Galdos, Carissa Lee, Soah Lee, Sharon Paige, William Goodyer, Sidra Xu, Tahmina Samad, Gabriela V. Escobar, Adrija Darsha, Aimee Beck, Rasmus O. Bak, Matthew H. Porteus, Sean M. Wu
Summary: The study provides a detailed characterization of human-induced pluripotent stem cells (hiPSCs) undergoing cardiac differentiation using genetic lineage tracing and single-cell transcriptomics. By comparing data from different model systems, they confirmed the predominance of left ventricular cardiomyocytes in hiPSC-derived progeny.
Article
Medicine, Research & Experimental
Nanna S. Mikkelsen, Sabina S. Hernandez, Trine I. Jensen, Jessica L. Schneller, Rasmus O. Bak
Summary: This article investigates a CRISPRa-induced enrichment strategy for transgenic cells, which can achieve higher editing efficiency in primary cells and promote the development of gene and cellular therapies. The strategy was successfully applied in human T cells and CD34+ hematopoietic stem and progenitor cells, resulting in a 2.5-fold enrichment of CAR T cells and improved cytotoxicity.
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
(2023)
Article
Oncology
Justin V. Joseph, Mathilde S. Blaavand, Huiqiang Cai, Fabienne Vernejoul, Rasmus W. Knopper, Thomas B. Lindhardt, Kristian A. Skipper, Esben Axelgaard, Line Reinert, Jacob G. Mikkelsen, Per Borghammer, Soren E. Degn, Eric Perouzel, Henrik Hager, Brian Hansen, Joanna M. Kalucka, Mikkel Vendelbo, Soren R. Paludan, Martin K. Thomsen
Summary: This study focused on glioblastoma and investigated the activation of the innate immune system through long-term STING activation as a potential treatment approach. The results showed that this approach reduced tumor progression and prolonged survival. Additionally, prolonged STING activation led to alterations in tumor vasculature and activation of VEGFR. Combination treatment with anti-PD1 did not provide any additional benefits, suggesting that STING activation alone is sufficient to hinder tumor development.
Article
Biochemistry & Molecular Biology
Fanghui Ren, Ryo Narita, Ahmad S. Rashidi, Stefanie Fruhwurth, Zongliang Gao, Rasmus O. Bak, Martin K. Thomsen, Georges M. G. M. Verjans, Line S. Reinert, Soren R. Paludan
Summary: Neurotropic viruses such as herpes simplex virus (HSV) can infect neurons and cause severe diseases. HSV-induced neuronal cell death is mediated by gasdermin E (GSDME) and involves endoplasmic reticulum stress, caspase activation, and mitochondria-dependent apoptosis. The necrotic neurons release alarmins, triggering inflammatory responses in microglia.
Article
Oncology
Signe Neldeborg, Johannes Frasez Soerensen, Charlotte Thornild Moller, Marie Bill, Zongliang Gao, Rasmus O. Bak, Kasper Holm, Boe Sorensen, Mette Nyegaard, Yonglun Luo, Peter Hokland, Magnus Stougaard, Maja Ludvigsen, Christian Kanstrup Holm
Summary: Oncogenic fusion drivers in hematological cancers can be targeted using a new dual intron-targeting CRISPR-Cas9 treatment strategy. This strategy can efficiently disrupt fusion genes without requiring precise knowledge of the breakpoints in t(8;21) AML. In vitro and in vivo experiments showed significant reduction in cell growth and tumor growth in response to disruption of RUNX1-RUNX1T1. These findings were confirmed in primary cells from an AML patient.
Article
Biochemistry & Molecular Biology
Jakob Haldrup, Sofie Andersen, Alexander Rafael LaVilla Labial, Jonas Holst Wolff, Frederik Plum Frandsen, Thomas Wisbech Skov, Anne Bruun Rovsing, Ian Nielsen, Thomas Stax Jakobsen, Anne Louise Askou, Martin K. Thomsen, Thomas J. Corydon, Emil Aagaard Thomsen, Jacob Giehm Mikkelsen
Summary: Effective delivery of gene editing tools in therapeutic in vivo gene editing using CRISPR/Cas can be achieved by engineering lentivirus-derived nanoparticles (LVNPs) as carriers for Cas protein and single guide RNA (sgRNA). LVNPs facilitate precise and efficient gene editing with reduced off-target cleavage activity, making them promising vehicles for in vivo gene disruption. The proof-of-concept study in mice demonstrates the potential of LVNPs for donor-free base and prime editing without double-stranded DNA breaks.
NUCLEIC ACIDS RESEARCH
(2023)
Review
Medicine, Research & Experimental
Sofie R. Dorset, Rasmus O. Bak
Summary: Ex vivo gene editing in hematopoietic stem and progenitor cells holds promise for treating monogenic blood disorders, but challenges remain regarding DNA damage response and p53 activation, which need further research for safe and efficient clinical implementation of gene editing.
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
(2023)
Article
Biochemistry & Molecular Biology
Troels Boldt Romer, Fawzi Khoder-Agha, Mikkel Koed Moller Aasted, Noortje de Haan, Sabrina Horn, August Dylander, Tao Zhang, Emil Marek Heymans Pallesen, Sally Dabelsteen, Manfred Wuhrer, Christine Flodgaard Hogsbro, Emil Aagaard Thomsen, Jacob Giehm Mikkelsen, Hans H. Wandall
Summary: This study reveals the mechanism behind the regulation of cancer-associated O-glycan expression by ZIP9 and zinc ions. Deletion of the ZIP9 gene or accumulation of zinc ions leads to cancer-like glycosylation, while overexpression of COSMC mitigates these changes.