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Journal articles on the topic 'DNA-free gene editing'

Author: Grafiati

Published: 11 March 2023

Last updated: 31 July 2025

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1

Haas, Amanda. "DNA-Free CRISPR-Cas9 Gene Editing." Genetic Engineering & Biotechnology News 36, no. 17 (2016): 16–17. http://dx.doi.org/10.1089/gen.36.17.07.

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Zhang, Yi, and Caixia Gao. "Recent advances in DNA-free editing and precise base editing in plants." Emerging Topics in Life Sciences 1, no. 2 (2017): 161–68. http://dx.doi.org/10.1042/etls20170021.

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Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly

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He, Yubing, Michael Mudgett, and Yunde Zhao. "Advances in gene editing without residual transgenes in plants." Plant Physiology 188, no. 4 (2021): 1757–68. http://dx.doi.org/10.1093/plphys/kiab574.

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Abstract Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently edit target genes without leaving any transgenes in plants. Some approaches directly address this issue by editing plant genomes with DNA-free reagents. On the other hand, DNA-based techniques require another step for ensuring plants are transgene-free. Fluorescent markers, pigments, and chemical treatments have all been employed as tools to distinguish transgenic plants from transgene-free plants quickly and easily.

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Tsanova, Tsveta, Lidia Stefanova, Lora Topalova, Atanas Atanasov, and Ivelin Pantchev. "DNA-free gene editing in plants: a brief overview." Biotechnology & Biotechnological Equipment 35, no. 1 (2020): 131–38. http://dx.doi.org/10.1080/13102818.2020.1858159.

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5

Brandt, Camilla Blunk, Sofie Vestergaard Fonager, János Haskó, et al. "HIF1A Knockout by Biallelic and Selection-Free CRISPR Gene Editing in Human Primary Endothelial Cells with Ribonucleoprotein Complexes." Biomolecules 13, no. 1 (2022): 23. http://dx.doi.org/10.3390/biom13010023.

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Primary endothelial cells (ECs), especially human umbilical vein endothelial cells (HUVECs), are broadly used in vascular biology. Gene editing of primary endothelial cells is known to be challenging, due to the low DNA transfection efficiency and the limited proliferation capacity of ECs. We report the establishment of a highly efficient and selection-free CRISPR gene editing approach for primary endothelial cells (HUVECs) with ribonucleoprotein (RNP) complex. We first optimized an efficient and cost-effective protocol for messenger RNA (mRNA) delivery into primary HUVECs by nucleofection. Ne

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Nasri, Masoud, Perihan Mir, Benjamin Dannenmann, et al. "Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response." Blood Advances 3, no. 1 (2019): 63–71. http://dx.doi.org/10.1182/bloodadvances.2017015511.

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Abstract CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9–gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for g

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Mushtaq, Muntazir, Aejaz Ahmad Dar, Milan Skalicky, et al. "CRISPR-Based Genome Editing Tools: Insights into Technological Breakthroughs and Future Challenges." Genes 12, no. 6 (2021): 797. http://dx.doi.org/10.3390/genes12060797.

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Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the nota

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Wang, Chengkun, Yuanhao Qu, Jason K. W. Cheng, et al. "dCas9-based gene editing for cleavage-free genomic knock-in of long sequences." Nature Cell Biology 24, no. 2 (2022): 268–78. http://dx.doi.org/10.1038/s41556-021-00836-1.

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AbstractGene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR–Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9–SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9–SSAP editor has low on-target errors and minimal off-target effects, showing h

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Badhan, Sapna, Andrew S. Ball, and Nitin Mantri. "First Report of CRISPR/Cas9 Mediated DNA-Free Editing of 4CL and RVE7 Genes in Chickpea Protoplasts." International Journal of Molecular Sciences 22, no. 1 (2021): 396. http://dx.doi.org/10.3390/ijms22010396.

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The current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome data, CRISPR/Cas9 presents a massive opportunity for plant breeders and researchers. The successful delivery of ribonucleoproteins (RNPs), which are composed of Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) and are used in combination with various transformation methods or lately av

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Ariga, Hirotaka, Seiichi Toki, and Kazuhiro Ishibashi. "Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants." Plant and Cell Physiology 61, no. 11 (2020): 1946–53. http://dx.doi.org/10.1093/pcp/pcaa123.

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Abstract Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, &gt;60% of shoot

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Sant’Ana, Rodrigo Ribeiro Arnt, Clarissa Alves Caprestano, Rubens Onofre Nodari, and Sarah Zanon Agapito-Tenfen. "PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts." Genes 11, no. 9 (2020): 1029. http://dx.doi.org/10.3390/genes11091029.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of

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Klimek-Chodacka, Magdalena, Miron Gieniec, and Rafal Baranski. "Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts." International Journal of Molecular Sciences 22, no. 19 (2021): 10740. http://dx.doi.org/10.3390/ijms221910740.

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The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or thre

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Zhang, Chao, Shanhe Liu, Xuan Li, Ruixuan Zhang, and Jun Li. "Virus-Induced Gene Editing and Its Applications in Plants." International Journal of Molecular Sciences 23, no. 18 (2022): 10202. http://dx.doi.org/10.3390/ijms231810202.

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CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generati

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Ferenczi, Aron, Douglas Euan Pyott, Andromachi Xipnitou, and Attila Molnar. "Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA." Proceedings of the National Academy of Sciences 114, no. 51 (2017): 13567–72. http://dx.doi.org/10.1073/pnas.1710597114.

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The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as ∼10% efficiency in C. reinhardtii. We demonstrate its use in transgene- and selection-free generation of sequence-specific mutations and epitope tagging a

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Chen, Xiaoyu, Francesca Tasca, Qian Wang, et al. "Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking." Nucleic Acids Research 48, no. 2 (2019): 974–95. http://dx.doi.org/10.1093/nar/gkz1121.

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Abstract Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR–Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at all

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Bollen, Yannik, Joris H. Hageman, Petra van Leenen, et al. "Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage." PLOS Biology 20, no. 1 (2022): e3001527. http://dx.doi.org/10.1371/journal.pbio.3001527.

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CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indel

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Xiu, Kemao, Laura Saunders, Luan Wen, et al. "Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing." Cells 12, no. 1 (2022): 156. http://dx.doi.org/10.3390/cells12010156.

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Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plas

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Chen, Kunling, Yanpeng Wang, Rui Zhang, Huawei Zhang, and Caixia Gao. "CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture." Annual Review of Plant Biology 70, no. 1 (2019): 667–97. http://dx.doi.org/10.1146/annurev-arplant-050718-100049.

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Enhanced agricultural production through innovative breeding technology is urgently needed to increase access to nutritious foods worldwide. Recent advances in CRISPR/Cas genome editing enable efficient targeted modification in most crops, thus promising to accelerate crop improvement. Here, we review advances in CRISPR/Cas9 and its variants and examine their applications in plant genome editing and related manipulations. We highlight base-editing tools that enable targeted nucleotide substitutions and describe the various delivery systems, particularly DNA-free methods, that have linked genom

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Kang, Beum-Chang, Su-Ji Bae, Seonghyun Lee, et al. "Chloroplast and mitochondrial DNA editing in plants." Nature Plants 7, no. 7 (2021): 899–905. http://dx.doi.org/10.1038/s41477-021-00943-9.

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AbstractPlant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector s

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Turner, Bryan. "Epigenetics can free us from the tyranny of selfish DNA." Biochemist 39, no. 5 (2017): 4–7. http://dx.doi.org/10.1042/bio03905004.

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Richard Dawkins' The Selfish Gene was published in 1976. It promulgates a philosophy that places DNA firmly in charge; you are what your DNA (acting in its own evolutionary interests) tells you to be. We know of course that things are not so simple, but the enthusiasm that greets scientific advances in gene editing may reflect a subliminal wish to escape the implacable determinism of DNA. Biochemistry shows us how the environment influences genome function by acting on nucleosomes and their component histones. But, to build on this, we need to know to what extent environmentally induced change

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Yan, Fang, Jingwen Wang, Sujie Zhang, et al. "CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates." PLOS Pathogens 19, no. 1 (2023): e1010961. http://dx.doi.org/10.1371/journal.ppat.1010961.

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CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled

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Namo, Fekede Meshesha. "Optimizing Genome Editing for Wheat Genetic Improvement: Current Status and Future Prospective." Bioscience Biotechnology Research Communications 16, no. 4 (2023): 212–18. https://doi.org/10.21786/bbrc/16.4.1.

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Wheat (Triticum aestivum L) is the main source of nutrition and feeds more than 30% of the world’s population. It is grown in various environments, providing most humans with around 20% of their calories and protein. Genome editing holds countless promise to accelerate the development of improved crop varieties including wheat via providing powerful tools to modify the genomic regions that controlling major agronomic traits. CRISPR/Cas9 system is an effective method for targeted genome editing. CRISPR/Cas9 allows researchers to target multiple homoeoalleles simultaneously and it enables the pr

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Kang, Minjeong, Keunsub Lee, Qing Ji, Sehiza Grosic, and Kan Wang. "Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector." Plants 12, no. 15 (2023): 2799. http://dx.doi.org/10.3390/plants12152799.

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Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assi

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Kim, Daesik, Kevin Luk, Scot A. Wolfe, and Jin-Soo Kim. "Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases." Annual Review of Biochemistry 88, no. 1 (2019): 191–220. http://dx.doi.org/10.1146/annurev-biochem-013118-111730.

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Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearran

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Lin, Choun-Sea, Chen-Tran Hsu, Yu-Hsuan Yuan, et al. "DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration." Plant Physiology 188, no. 4 (2022): 1917–30. http://dx.doi.org/10.1093/plphys/kiac022.

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Abstract Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA

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Nascimento, Fernanda dos Santos, Anelita de Jesus Rocha, Julianna Matos da Silva Soares, et al. "Gene Editing for Plant Resistance to Abiotic Factors: A Systematic Review." Plants 12, no. 2 (2023): 305. http://dx.doi.org/10.3390/plants12020305.

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Agricultural crops are exposed to various abiotic stresses, such as salinity, water deficits, temperature extremes, floods, radiation, and metal toxicity. To overcome these challenges, breeding programs seek to improve methods and techniques. Gene editing by Clustered Regularly Interspaced Short Palindromic Repeats—CRISPR/Cas—is a versatile tool for editing in all layers of the central dogma with focus on the development of cultivars of plants resistant or tolerant to multiple biotic or abiotic stresses. This systematic review (SR) brings new contributions to the study of the use of CRISPR/Cas

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T, Ugandhar, Komala E, Shyam Prasad. S, et al. "Revolutionizing plant breeding: a historical perspective from cross-pollination to gene editing." International Journal of Biological Research 11, no. 2 (2024): 57–66. http://dx.doi.org/10.14419/2sz7ka92.

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The history of plant breeding can be broadly divided into four eras: the Pre-Mendelian era (before 1900), the Mendelian era (1900-1920), the post-Mendelian era (1921-1950), and the Modern era (after 1950). Significant milestones include the release of the first hybrid maize varieties in 1961, the first sorghum hybrid (CSH-1) in 1964, the first bajra hybrid (HB-1) in 1965, and the first pigeon pea hybrid (ICPH-8) in 1991. Achieving a world with zero hunger requires a sustainable increase in food production and distribution and elimination of poverty. This goal demands scientific, logistical, an

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Veillet, Florian, Laura Perrot, Laura Chauvin, et al. "Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor." International Journal of Molecular Sciences 20, no. 2 (2019): 402. http://dx.doi.org/10.3390/ijms20020402.

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Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, esp

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Zegeye, Workie Anley, Mesfin Tsegaw, Yingxin Zhang, and Liyong Cao. "CRISPR-Based Genome Editing: Advancements and Opportunities for Rice Improvement." International Journal of Molecular Sciences 23, no. 8 (2022): 4454. http://dx.doi.org/10.3390/ijms23084454.

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To increase the potentiality of crop production for future food security, new technologies for plant breeding are required, including genome editing technology—being one of the most promising. Genome editing with the CRISPR/Cas system has attracted researchers in the last decade as a safer and easier tool for genome editing in a variety of living organisms including rice. Genome editing has transformed agriculture by reducing biotic and abiotic stresses and increasing yield. Recently, genome editing technologies have been developed quickly in order to avoid the challenges that genetically modi

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Asaoka, Mariko, Takashi Ishikawa, Kazuaki Takabe, and Santosh K. Patnaik. "APOBEC3-Mediated RNA Editing in Breast Cancer is Associated with Heightened Immune Activity and Improved Survival." International Journal of Molecular Sciences 20, no. 22 (2019): 5621. http://dx.doi.org/10.3390/ijms20225621.

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APOBEC3 enzymes contribute significantly to DNA mutagenesis in cancer. These enzymes are also capable of converting C bases at specific positions of RNAs to U. However, the prevalence and significance of this C-to-U RNA editing in any cancer is currently unknown. We developed a bioinformatics workflow to determine RNA editing levels at known APOBEC3-mediated RNA editing sites using exome and mRNA sequencing data of 1040 breast cancer tumors. Although reliable editing determinations were limited due to sequencing depth, editing was observed in both tumor and adjacent normal tissues. For 440 sit

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Ryu, J., W. Chan, F. Carvalho, E. Mishler, J. Hennebold, and C. Hanna. "109 Utilising cell-free DNA for detection of gene editing outcomes in rhesus macaque embryos." Reproduction, Fertility and Development 34, no. 2 (2022): 291. http://dx.doi.org/10.1071/rdv34n2ab109.

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Schnütgen, Frank, Duran Sürün, Joachim Schwäble, et al. "High Efficiency Gene Correction in Hematopoietic Cells By Template-Free Crispr/Cas9 Genome Editing." Blood 128, no. 22 (2016): 3507. http://dx.doi.org/10.1182/blood.v128.22.3507.3507.

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Abstract Introduction A significant fraction of inherited monogenic disorders is caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical gene therapy trials. Most of these trials employed retroviral vectors, which by inserting randomly throughout the genome deprived the transduced genes of their endogenous control and caused insertional mutations leading to secondary disease. The development of genome editing tools capable

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Shin, Yun-Hee, and Young-Doo Park. "CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)." International Journal of Molecular Sciences 24, no. 1 (2022): 541. http://dx.doi.org/10.3390/ijms24010541.

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Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the ‘CT001’ pseudomolecule, a single guide RNA (sgRNA) was designe

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Pavese, Vera, Andrea Moglia, Silvia Abbà, et al. "First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill." International Journal of Molecular Sciences 23, no. 10 (2022): 5762. http://dx.doi.org/10.3390/ijms23105762.

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Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous

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Mikl, Marie C., Ian N. Watt, Mason Lu, et al. "Mice Deficient in APOBEC2 and APOBEC3." Molecular and Cellular Biology 25, no. 16 (2005): 7270–77. http://dx.doi.org/10.1128/mcb.25.16.7270-7277.2005.

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ABSTRACT The activation-induced deaminase/apolipoprotein B-editing catalytic subunit 1 (AID/APOBEC) family comprises four groups of proteins. Both AID, a lymphoid-specific DNA deaminase that triggers antibody diversification, and APOBEC2 (function unknown) are found in all vertebrates examined. In contrast, APOBEC1, an RNA-editing enzyme in gastrointestinal cells, and APOBEC3 are restricted to mammals. The function of most APOBEC3s, of which there are seven in human but one in mouse, is unknown, although several human APOBEC3s act as host restriction factors that deaminate human immunodeficien

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Hewes, Amanda M., Brett M. Sansbury, and Eric B. Kmiec. "The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template." Genes 11, no. 10 (2020): 1160. http://dx.doi.org/10.3390/genes11101160.

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correct

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Hoengenaert, Lennart, Chantal Anders, Jan Van Doorsselaere, Ruben Vanholme, and Wout Boerjan. "Transgene‐free genome editing in poplar." New Phytologist, January 22, 2025. https://doi.org/10.1111/nph.20415.

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Summary Precise gene‐editing methods are valuable tools to enhance genetic traits. Gene editing is commonly achieved via stable integration of a gene‐editing cassette in the plant's genome. However, this technique is unfavorable for field applications, especially in vegetatively propagated plants, such as many commercial tree species, where the gene‐editing cassette cannot be segregated away without breaking the genetic constitution of the elite variety. Here, we describe an efficient method for generating gene‐edited Populus tremula × P. alba (poplar) trees without incorporating foreign DNA i

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Van den Broeck, Senne, Yvan Ngapout, Bart Panis, and Hervé Vanderschuren. "An Agrobacterium‐mediated base editing approach generates transgene‐free edited banana." New Phytologist, April 2025. https://doi.org/10.1111/nph.70044.

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Summary Genome editing for the development of improved varieties is supported by the possibility of segregating out the editor T‐DNA cassette after genome editing in many crop species. Removal of the T‐DNA cassette prevents potential continuous editing activity in the transformed plant and furthermore facilitates regulatory approval. While transgene outcrossing of exogenous sequences is possible for many crops, this is not the case for vegetatively propagated and sterile crops, such as Cavendish bananas. Therefore, gene editing techniques leading to transgene‐free edited plants are essential t

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Zhang, Xin, Xinlong Wang, Jie Lv, et al. "Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing." Genome Biology 24, no. 1 (2023). http://dx.doi.org/10.1186/s13059-023-02992-z.

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Abstract Background The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability. Results Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximate

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Creeth, Ryan, Andrew Thompson, and Zoltan Kevei. "DNA-free CRISPR genome editing in raspberry (Rubus idaeus) protoplast through RNP-mediated transfection." Frontiers in Genome Editing 7 (June 30, 2025). https://doi.org/10.3389/fgeed.2025.1589431.

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Protoplast-based systems have been utilised in a wide variety of plant species to enable genome editing without chromosomal introgression of foreign DNA into plant genomes. DNA-free genome editing followed by protoplast regeneration allows elite cultivars to be edited without further genetic segregation, preserving their unique genetic composition and their regulatory status as non-transgenic. However, protoplast isolation presents a barrier to the development of advanced breeding technologies in raspberry and no protocol has been published for DNA-free genome editing in the species. Pre-assem

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Hoengenaert, Lennart, Jan Van Doorsselaere, Ruben Vanholme, and Wout Boerjan. "Microparticle-mediated CRISPR DNA delivery for genome editing in poplar." Frontiers in Plant Science 14 (November 13, 2023). http://dx.doi.org/10.3389/fpls.2023.1286663.

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The use of CRISPR/Cas9 is currently the method of choice for precise genome engineering in plants, including in the biomass crop poplar. The most commonly used method for delivering CRISPR/Cas9 and its components in poplar is via Agrobacterium-mediated transformation, that besides the desired gene-editing event also results in stable T-DNA integration. Here we explore the delivery of the gene-editing reagents via DNA-coated microparticle bombardment into the model tree Populus tremula x P. alba to evaluate its potential for developing transgene-free, gene-edited trees, as well as its potential

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Teufel, Marc, Carlo A. Klein, Maurice Mager, and Patrick Sobetzko. "A multifunctional system for genome editing and large-scale interspecies gene transfer." Nature Communications 13, no. 1 (2022). http://dx.doi.org/10.1038/s41467-022-30843-1.

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AbstractCRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. We demonstrate the excision, transfer and integration of large chromosomal regions between E. coli, V. natriegens and D. dadantii without size-limiting intermediate DNA

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Kim, Yunsun, Eunbin Lee, and Beum‐Chang Kang. "Etiolation promotes protoplast transfection and genome editing efficiency." Physiologia Plantarum 176, no. 6 (2024). http://dx.doi.org/10.1111/ppl.14637.

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AbstractIn plants, DNA‐free genome editing using preassembled clustered regularly interspaced short palindromic repeats (CRISPR)‐ribonucleoprotein (RNP) has the advantage of avoiding transgene integration and limiting off‐target effects. The efficiency of this gene editing strategy can vary, so optimization of protoplast transfection conditions is necessary to achieve maximum yield. In this study, we examined the effects of etiolation, or increased exposure to darkness during cultivation, on the transfection efficiency of protoplasts from lettuce and Chinese cabbage. Seedlings were grown under

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Chen, Yu-Hao, Jun Lu, Xia Yang, et al. "Gene editing of non-coding regulatory DNA and its application in crop improvement." Journal of Experimental Botany, August 7, 2023. http://dx.doi.org/10.1093/jxb/erad313.

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Abstract The development of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has provided precise and efficient strategies to edit target genes and generate transgene-free crops. Significant progress has been made in the editing of protein coding genes; however, studies on the editing of non-coding DNA with regulatory roles lags far behind. Non-coding regulatory DNAs, including long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and cis-regulatory elements (CREs), play crucial roles in regulating plant growth and development. Therefore,

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Shih, Sra-Yh, Uffe Hasbro Mortensen, Fang-Rong Chang, and HsinYuan Tsai. "Editing Aspergillus terreus using the CRISPR-Cas9 system." Synthetic Biology, December 6, 2022. http://dx.doi.org/10.1093/synbio/ysac031.

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Abstract CRISPR-Cas9 technology has been utilized in different organisms for targeted mutagenesis, offering a fast, precise, and cheap approach to speed up molecular breeding and study of gene function. Until now, many researchers have established the demonstration of applying the CRISPR/Cas9 system in various fungal model species. However, there are very few guidelines available for CRISPR/Cas9 genome editing in Aspergillus terreus. In this study, we present CRISPR/Cas9 genome editing in A. terreus. To optimize the gRNA expression, we constructed a modified sgRNA/Cas9 expression plasmid. By c

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Chen, Hao, Matthew Neubauer, and Jack P. Wang. "Enhancing HR Frequency for Precise Genome Editing in Plants." Frontiers in Plant Science 13 (May 3, 2022). http://dx.doi.org/10.3389/fpls.2022.883421.

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Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene ed

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Poddar, Snigdha, Jaclyn Tanaka, Katherine L. D. Running, et al. "Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.)." Frontiers in Plant Science 13 (January 10, 2023). http://dx.doi.org/10.3389/fpls.2022.1084700.

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The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assemb

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Yue, Jin-Jun, Jin-Ling Yuan, Fu-Hui Wu, et al. "Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application." Frontiers in Genome Editing 3 (August 11, 2021). http://dx.doi.org/10.3389/fgeed.2021.717017.

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In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report,

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Huang, Xiaoen, Yuanchun Wang, and Nian Wang. "Base Editors for Citrus Gene Editing." Frontiers in Genome Editing 4 (February 28, 2022). http://dx.doi.org/10.3389/fgeed.2022.852867.

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Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to t

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Laforest, Larissa C., and Satya Swathi Nadakuduti. "Advances in Delivery Mechanisms of CRISPR Gene-Editing Reagents in Plants." Frontiers in Genome Editing 4 (January 24, 2022). http://dx.doi.org/10.3389/fgeed.2022.830178.

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Gene-editing by CRISPR/Cas systems has revolutionized plant biology by serving as a functional genomics tool. It has tremendously advanced plant breeding and crop improvement by accelerating the development of improved cultivars, creating genetic variability, and aiding in domestication of wild and orphan crops. Gene-editing is a rapidly evolving field. Several advancements include development of different Cas effectors with increased target range, efficacy, and enhanced capacity for precise DNA modifications with base editing and prime editing. The existing toolbox of various CRISPR reagents

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