Статті в журналах: "Base editors"

1

Cao, Di, Manzar Abbas, Fatima, Yunxia Li, Gaoping Zhao, Ghulam Abbas, and Xihe Li. "BASE EDITING: A PROMISING TOOL FOR GENETIC MANIPULATION IN MAMMALIAN SOMATIC AND STEM CELL LINES." Pakistan Journal of Science 76, no. 04 (December 30, 2024): 553–69. https://doi.org/10.57041/vol76iss04pp553-569.

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The CRISPR system enables precise editing in genomic DNA but relies on intracellular homology-directed recombination (HDR) repair pathways and is extremely inefficient. Base editing technology developed based on the CRISPR/Cas9 system builds three Base editors (BE) by fusing nucleases that have lost cutting activity with different base deaminases: Cytosine base editor (CBE) and Adenine base editor (ABE) and Glycosylase base editors (GBE). These two types of editors can complete the substitution of C > T (G > A) or A > G (T > C) at gene target sites without producing DNA double-strand breaks, and finally achieve accurate base editing. At present, base editing technology has been widely used in gene therapy, animal model construction, precision animal breeding, gene function analysis, and other fields, providing a powerful technical tool for basic and applied research. This paper summarizes the development and optimization process of base editing technology, and its application in livestock and poultry, to provide a reference for researchers in related fields to use base editing systems.

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2

Bellingrath, Julia-Sophia, Michelle E. McClements, Maria Kaukonen, Manuel Dominik Fischer, and Robert E. MacLaren. "In Silico Analysis of Pathogenic CRB1 Single Nucleotide Variants and Their Amenability to Base Editing as a Potential Lead for Therapeutic Intervention." Genes 12, no. 12 (November 27, 2021): 1908. http://dx.doi.org/10.3390/genes12121908.

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Mutations in the Crumbs homolog 1 (CRB1) gene cause both autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Since three separate CRB1 isoforms are expressed at meaningful levels in the human retina, base editing shows promise as a therapeutic approach. This retrospective analysis aims to summarise the reported pathogenic CRB1 variants and investigate their amenability to treatment with currently available DNA base editors. Pathogenic single nucleotide variants (SNVs) were extracted from the Leiden open-source variation database (LOVD) and ClinVar database and coded by mutational consequence. They were then analyzed for their amenability to currently available DNA base editors and available PAM sites from a selection of different Cas proteins. Of a total of 1115 unique CRB1 variants, 69% were classified as pathogenic SNVs. Of these, 62% were amenable to currently available DNA BEs. Adenine base editors (ABEs) alone have the potential of targeting 34% of pathogenic SNVs; 19% were amenable to a CBE while GBEs could target an additional 9%. Of the pathogenic SNVs targetable with a DNA BE, 87% had a PAM site for a Cas protein. Of the 33 most frequently reported pathogenic SNVs, 70% were targetable with a base editor. The most common pathogenic variant was c.2843G>A, p.Cys948Arg, which is targetable with an ABE. Since 62% of pathogenic CRB1 SNVs are amenable to correction with a base editor and 87% of these mutations had a suitable PAM site, gene editing represents a promising therapeutic avenue for CRB1-associated retinal degenerations.

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3

Evanoff, Mallory, and Alexis C. Komor. "Base editors: modular tools for the introduction of point mutations in living cells." Emerging Topics in Life Sciences 3, no. 5 (September 10, 2019): 483–91. http://dx.doi.org/10.1042/etls20190088.

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Base editors are a new family of programmable genome editing tools that fuse ssDNA (single-stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.

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4

Monsur, Mahmuda Binte, Gaoneng Shao, Yusong Lv, Shakeel Ahmad, Xiangjin Wei, Peisong Hu, and Shaoqing Tang. "Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants." Genes 11, no. 4 (April 24, 2020): 466. http://dx.doi.org/10.3390/genes11040466.

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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach.

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5

Kantor, Ariel, Michelle McClements, and Robert MacLaren. "CRISPR-Cas9 DNA Base-Editing and Prime-Editing." International Journal of Molecular Sciences 21, no. 17 (August 28, 2020): 6240. http://dx.doi.org/10.3390/ijms21176240.

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Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.

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6

Rusk, Nicole. "Better base editors." Nature Methods 15, no. 10 (October 2018): 763. http://dx.doi.org/10.1038/s41592-018-0154-4.

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7

Tang, Lei. "Base editors beware." Nature Methods 17, no. 1 (January 2020): 21. http://dx.doi.org/10.1038/s41592-019-0705-3.

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8

Buchumenski, Ilana, Shalom Hillel Roth, Eli Kopel, Efrat Katsman, Ariel Feiglin, Erez Y. Levanon, and Eli Eisenberg. "Global quantification exposes abundant low-level off-target activity by base editors." Genome Research 31, no. 12 (October 19, 2021): 2354–61. http://dx.doi.org/10.1101/gr.275770.121.

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Base editors are dedicated engineered deaminases that enable directed conversion of specific bases in the genome or transcriptome in a precise and efficient manner, and hold promise for correcting pathogenic mutations. A major concern limiting application of this powerful approach is the issue of off-target edits. Several recent studies have shown substantial off-target RNA activity induced by base editors and demonstrated that off-target mutations may be suppressed by improved deaminases versions or optimized guide RNAs. Here, we describe a new class of off-target events that are invisible to the established methods for detection of genomic variations and were thus far overlooked. We show that nonspecific, seemingly stochastic, off-target events affect a large number of sites throughout the genome or the transcriptome, and account for the majority of off-target activity. We develop and employ a different, complementary approach that is sensitive to the stochastic off-target activity and use it to quantify the abundant off-target RNA mutations due to current, optimized deaminase editors. We provide a computational tool to quantify global off-target activity, which can be used to optimize future base editors. Engineered base editors enable directed manipulation of the genome or transcriptome at single-base resolution. We believe that implementation of this computational approach would facilitate design of more specific base editors.

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9

Aparicio-Prat, Estel, Dong Yan, Marco Mariotti, Michael Bassik, Gaelen Hess, Jean-Philippe Fortin, Andrea Weston, Hualin S. Xi, and Robert Stanton. "Roadmap for the use of base editors to decipher drug mechanism of action." PLOS ONE 16, no. 9 (September 21, 2021): e0257537. http://dx.doi.org/10.1371/journal.pone.0257537.

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CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The β2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.

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10

Li, Chang, Aphrodite Georgakopoulou, Arpit Mishra, Sucheol Gil, R. David Hawkins, Evangelia Yannaki та André Lieber. "In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice". Blood Advances 5, № 4 (23 лютого 2021): 1122–35. http://dx.doi.org/10.1182/bloodadvances.2020003702.

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Abstract Base editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over β-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the –113A>G HPFH mutation. In a β-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human β-globin. The average –113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage–negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of β-hemoglobinopathies.

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11

Miquel-Ribé, Marc. "User Engagement on Wikipedia, A Review of Studies of Readers and Editors." Proceedings of the International AAAI Conference on Web and Social Media 9, no. 5 (August 3, 2021): 67–74. http://dx.doi.org/10.1609/icwsm.v9i5.14695.

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Is it an encyclopedia or a social network? Without considering both aspects it would not be possible to understand how a worldwide army of editors created the largest online knowledge repository. Wikipedia has a consistent set of rules and it responds to many of the User Engagement Framework attributes, and this is why it works. In this paper, we identify these confirmed attributes as well as those presenting problems. We explain that although having a strong editor base Wikipedia is finding it challenging to maintain this base or increase its size. In order to understand this, scholars have analyzed Wikipedia using current metrics like user session and activity. We conclude there exist opportunities to analyze engagement in new aspects in order to understand its success, as well as to redesign mechanisms to improve the system and help the transition between reader and editor.

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12

Rabinowitz, Roy, Shiran Abadi, Shiri Almog, and Daniel Offen. "Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing." Nucleic Acids Research 48, W1 (April 7, 2020): W340—W347. http://dx.doi.org/10.1093/nar/gkaa215.

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Abstract Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A deaminase enzyme is fused to a deactivated Cas and enables transition conversions. The diversified repertoire of base editors provides a wide range of base editing possibilities. However, existing base editors cannot induce transversion substitutions and activate only within a specified region relative to the binding site, thus, they cannot precisely correct every point mutation. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a point mutation. When a precise correction is impossible, BE-FF aims to mutate bystander nucleotides in order to induce synonymous corrections that will correct the coding sequence. To measure BE-FF practicality, we analysed a database of human pathogenic point mutations. Out of the transition mutations, 60.9% coding sequences could be corrected. Notably, 19.4% of the feasible corrections were not achieved by precise corrections but only by synonymous corrections. Moreover, 298 cases of transversion-derived pathogenic mutations were detected to be potentially repairable by base editing via synonymous corrections, although base editing is considered impractical for such mutations.

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13

Miller, Marsha A., and Douglas N. Miller. "Early Journal Articles and Editors That Shaped the Evolution of Scholarly Writing in Academic Advising, 1972-2001." NACADA Review 3, no. 1 (January 1, 2022): 42–58. http://dx.doi.org/10.12930/nacr-21-19.

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This article provides a historical perspective on the development of scholarly writing in academic advising beginning in 1972 with some of the first journal articles solely devoted to advising and continuing through the initial 20 years of the NACADA Journal—the premier advising publication venue during the period. An important part of the evolution of advising's scholarly writing was the vision and perspective of each NACADA Journal editor. Each brought a distinct set of academic experiences to the job, each defined scholarship, and especially research, differently. Early (1972–2001) scholarly articles and the editors who published them shaped academic advising's literature base. This article analyzes the contributions made by these early articles and editors.

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14

Schneider, Robert C., and Jerzy Kosiewicz. "Robert Charles Schneider as a Proud Director of one of the First and Finest Higher Education Sport Management Programs in USA and World." Physical Culture and Sport. Studies and Research 76, no. 1 (December 1, 2017): 64–70. http://dx.doi.org/10.1515/pcssr-2017-0030.

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AbstractThis is the sixth article of the cycle of portraits of the members of the Editorial Board and Editorial Advisory Board of the journal Physical Culture and Sport. Studies and Research. These members are social scientists who research the issue of sport. Among them, there are many world-class professors, rectors, and deans of excellent universities, founders, presidents, and secretaries-general of continental and international scientific societies and editors of high-scoring journals related to social sciences focusing on sport. The idea of presenting portraits of individual editors of our writings has already gained recognition in the Far East. Editor-in-Chief Young Lee of the International Journal of Eastern Sports & Physical Education has decided to introduce Corner of Editors, which will also present all members of the Editorial Board.I would like to inform also that our Journal entitled Physical Culture and Sport. Studies and Research has been included into the base Emerging Sources Citation Index (ESCI), which is a part Web of Science (WoS). The Clarivate Analytics is the base that patronizes activity of the ESCI and WoS, and continue activity of Thomson Reuters.The biography we present here in this volume of our journal refers to a scholar from USA, educationist, and manager, Robert Charles Schneider.

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15

Rabaan, Ali A., Mona A. Al Fares, Manar Almaghaslah, Tariq Alpakistany, Nawal A. Al Kaabi, Saleh A. Alshamrani, Ahmad A. Alshehri, et al. "Application of CRISPR-Cas System to Mitigate Superbug Infections." Microorganisms 11, no. 10 (September 26, 2023): 2404. http://dx.doi.org/10.3390/microorganisms11102404.

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Multidrug resistance in bacterial strains known as superbugs is estimated to cause fatal infections worldwide. Migration and urbanization have resulted in overcrowding and inadequate sanitation, contributing to a high risk of superbug infections within and between different communities. The CRISPR-Cas system, mainly type II, has been projected as a robust tool to precisely edit drug-resistant bacterial genomes to combat antibiotic-resistant bacterial strains effectively. To entirely opt for its potential, advanced development in the CRISPR-Cas system is needed to reduce toxicity and promote efficacy in gene-editing applications. This might involve base-editing techniques used to produce point mutations. These methods employ designed Cas9 variations, such as the adenine base editor (ABE) and the cytidine base editor (CBE), to directly edit single base pairs without causing DSBs. The CBE and ABE could change a target base pair into a different one (for example, G-C to A-T or C-G to A-T). In this review, we addressed the limitations of the CRISPR/Cas system and explored strategies for circumventing these limitations by applying diverse base-editing techniques. Furthermore, we also discussed recent research showcasing the ability of base editors to eliminate drug-resistant microbes.

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16

Neff, Ellen P. "Base editors versus PKU." Lab Animal 48, no. 1 (December 12, 2018): 27. http://dx.doi.org/10.1038/s41684-018-0214-5.

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17

Burgess, Darren J. "Multitasking for base editors." Nature Reviews Genetics 21, no. 8 (June 23, 2020): 445. http://dx.doi.org/10.1038/s41576-020-0261-9.

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18

Li, Mengyuan, Yi-Xin Huo, and Shuyuan Guo. "CRISPR-Mediated Base Editing: From Precise Point Mutation to Genome-Wide Engineering in Nonmodel Microbes." Biology 11, no. 4 (April 9, 2022): 571. http://dx.doi.org/10.3390/biology11040571.

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Nonmodel microbes with unique and diverse metabolisms have become rising stars in synthetic biology; however, the lack of efficient gene engineering techniques still hinders their development. Recently, the use of base editors has emerged as a versatile method for gene engineering in a wide range of organisms including nonmodel microbes. This method is a fusion of impaired CRISPR/Cas9 nuclease and base deaminase, enabling the precise point mutation at the target without inducing homologous recombination. This review updates the latest advancement of base editors in microbes, including the conclusion of all microbes that have been researched by base editors, the introduction of newly developed base editors, and their applications. We provide a list that comprehensively concludes specific applications of BEs in nonmodel microbes, which play important roles in industrial, agricultural, and clinical fields. We also present some microbes in which BEs have not been fully established, in the hope that they are explored further and so that other microbial species can achieve arbitrary base conversions. The current obstacles facing BEs and solutions are put forward. Lastly, the highly efficient BEs and other developed versions for genome-wide reprogramming of cells are discussed, showing great potential for future engineering of nonmodel microbes.

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Lapinaite, Audrone, Gavin J. Knott, Cody M. Palumbo, Enrique Lin-Shiao, Michelle F. Richter, Kevin T. Zhao, Peter A. Beal, David R. Liu, and Jennifer A. Doudna. "DNA capture by a CRISPR-Cas9–guided adenine base editor." Science 369, no. 6503 (July 30, 2020): 566–71. http://dx.doi.org/10.1126/science.abb1390.

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CRISPR-Cas–guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo–electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA–like conformation. Furthermore, ABE8e’s accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

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Xiong, Xiangyu, Zhenxiang Li, Jieping Liang, Kehui Liu, Chenlong Li, and Jian-Feng Li. "A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants." Nucleic Acids Research 50, no. 6 (March 14, 2022): 3565–80. http://dx.doi.org/10.1093/nar/gkac166.

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Abstract CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.

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Hua, Kai, Peijin Han, and Jian-Kang Zhu. "Improvement of base editors and prime editors advances precision genome engineering in plants." Plant Physiology 188, no. 4 (December 28, 2021): 1795–810. http://dx.doi.org/10.1093/plphys/kiab591.

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Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-mediated gene disruption has revolutionized biomedical research as well as plant and animal breeding. However, most disease-causing mutations and agronomically important genetic variations are single base polymorphisms (single-nucleotide polymorphisms) that require precision genome editing tools for correction of the sequences. Although homology-directed repair of double-stranded breaks (DSBs) can introduce precise changes, such repairs are inefficient in differentiated animal and plant cells. Base editing and prime editing are two recently developed genome engineering approaches that can efficiently introduce precise edits into target sites without requirement of DSB formation or donor DNA templates. They have been applied in several plant species with promising results. Here, we review the extensive literature on improving the efficiency, target scope, and specificity of base editors and prime editors in plants. We also highlight recent progress on base editing in plant organellar genomes and discuss how these precision genome editing tools are advancing basic plant research and crop breeding.

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Pakari, Kaisa, Joachim Wittbrodt, and Thomas Thumberger. "CRISPR-Fortschritte — Schnitt für Schnitt zu neuen Möglichkeiten." BIOspektrum 29, no. 1 (February 2023): 25–28. http://dx.doi.org/10.1007/s12268-023-1893-z.

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AbstractCRISPR/Cas9 systems and recently established base editors are essential tools for precise, targeted genome editing for translational and basic research applications. Here we present small, easily combined improvements to reach editing versatility and enhanced efficiency. This is achieved by improved nuclear localization of Cas9, protected DNA sequences for homology directed repair and a combinatorial use of base editors to reach initially inaccessible target sites.

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Pallaseni, Ananth, Elin Madli Peets, Jonas Koeppel, Juliane Weller, Thomas Vanderstichele, Uyen Linh Ho, Luca Crepaldi, Jolanda van Leeuwen, Felicity Allen, and Leopold Parts. "Predicting base editing outcomes using position-specific sequence determinants." Nucleic Acids Research 50, no. 6 (March 14, 2022): 3551–64. http://dx.doi.org/10.1093/nar/gkac161.

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Abstract CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window. The impact of features on editing rate depends on the position, with sequence bias efficacy mainly influencing bases away from the center of the window. We use these observations to train a machine learning model to predict editing activity per position, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of disease mutation correcting guides, and find that most of them suffer from more unwanted editing than pure outcomes. This work unravels the position-specificity of base editing biases and allows more efficient planning of editing campaigns in experimental and therapeutic contexts.

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Liang, Mingming, Tingting Sui, Zhiquan Liu, Mao Chen, Hongmei Liu, Huanhuan Shan, Liangxue Lai, and Zhanjun Li. "AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects." Cells 9, no. 8 (July 27, 2020): 1786. http://dx.doi.org/10.3390/cells9081786.

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The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly “shut off” nuclease activity of both CBE and ABE systems.

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Waterbury, Amanda L., Irtiza Iram, and Brian B. Liau. "Building the first base editors." Nature Chemical Biology 21, no. 1 (December 24, 2024): 16–17. https://doi.org/10.1038/s41589-024-01790-3.

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O’Leary, Karen. "Base editors in the clinic." Nature Medicine 29, no. 12 (December 2023): 2972. http://dx.doi.org/10.1038/s41591-023-02708-7.

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Kaukonen, Maria, Michelle E. McClements, and Robert E. MacLaren. "CRISPR DNA Base Editing Strategies for Treating Retinitis Pigmentosa Caused by Mutations in Rhodopsin." Genes 13, no. 8 (July 26, 2022): 1327. http://dx.doi.org/10.3390/genes13081327.

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Анотація:

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerations and pathogenic variants in the Rhodopsin (RHO) gene are major cause for autosomal dominant RP (adRP). Despite extensive attempts to treat RHO-associated adRP, standardized curative treatment is still lacking. Recently developed base editors offer an exciting opportunity to correct pathogenic single nucleotide variants and are currently able to correct all transition variants and some transversion variants. In this study, we analyzed previously reported pathogenic RHO variants (n = 247) for suitable PAM sites for currently available base editors utilizing the Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9) or the KKH variant of SaCas9 (KKH-SaCas9) to assess DNA base editing as a treatment option for RHO-associated adRP. As a result, 55% of all the analyzed variants could, in theory, be corrected with base editors, however, PAM sites were available for only 32% of them and unwanted bystander edits were predicted for the majority of the designed guide RNAs. As a conclusion, base editing offers exciting possibilities to treat RHO-associated adRP in the future, but further research is needed to develop base editing constructs that will provide available PAM sites for more variants and that will not introduce potentially harmful bystander edits.

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Seidel Malkinson, Tal, Devin B. Terhune, Mathew Kollamkulam, Maria J. Guerreiro, Dani S. Bassett, and Tamar R. Makin. "Gender imbalances in the editorial activities of a selective journal run by academic editors." PLOS ONE 18, no. 12 (December 11, 2023): e0294805. http://dx.doi.org/10.1371/journal.pone.0294805.

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The fairness of decisions made at various stages of the publication process is an important topic in meta-research. Here, based on an analysis of data on the gender of authors, editors and reviewers for 23,876 initial submissions and 7,192 full submissions to the journal eLife, we report on five stages of the publication process. We find that the board of reviewing editors (BRE) is men-dominant (69%) and that authors disproportionately suggest male editors when making an initial submission. We do not find evidence for gender bias when Senior Editors consult Reviewing Editors about initial submissions, but women Reviewing Editors are less engaged in discussions about these submissions than expected by their proportion. We find evidence of gender homophily when Senior Editors assign full submissions to Reviewing Editors (i.e., men are more likely to assign full submissions to other men (77% compared to the base assignment rate to men RE of 70%), and likewise for women (41% compared to women RE base assignment rate of 30%))). This tendency was stronger in more gender-balanced scientific disciplines. However, we do not find evidence for gender bias when authors appeal decisions made by editors to reject submissions. Together, our findings confirm that gender disparities exist along the editorial process and suggest that merely increasing the proportion of women might not be sufficient to eliminate this bias. Measures accounting for women’s circumstances and needs (e.g., delaying discussions until all RE are engaged) and raising editorial awareness to women’s needs may be essential to increasing gender equity and enhancing academic publication.

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Chen, Liwei, Jung Eun Park, Peter Paa, Priscilla D. Rajakumar, Hong-Ting Prekop, Yi Ting Chew, Swathi N. Manivannan, and Wei Leong Chew. "Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins." Nature Communications 12, no. 1 (March 2, 2021). http://dx.doi.org/10.1038/s41467-021-21559-9.

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Анотація:

AbstractMany genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.

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30

Chen, Fangbing, Meng Lian, Bingxiu Ma, Shixue Gou, Xian Luo, Kaiming Yang, Hui Shi, et al. "Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors." Communications Biology 5, no. 1 (November 2, 2022). http://dx.doi.org/10.1038/s42003-022-04152-8.

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Анотація:

AbstractCas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.

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Tong, Huawei, Haoqiang Wang, Xuchen Wang, Nana Liu, Guoling Li, Danni Wu, Yun Li, et al. "Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase." Nature Communications 15, no. 1 (June 8, 2024). http://dx.doi.org/10.1038/s41467-024-49343-5.

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Анотація:

AbstractDNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

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32

Kweon, Jiyeon, An-Hee Jang, Eunji Kwon, Ungi Kim, Ha Rim Shin, Jieun See, Gayoung Jang, et al. "Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery." Experimental & Molecular Medicine, February 1, 2023. http://dx.doi.org/10.1038/s12276-023-00938-w.

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AbstractVarious CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.

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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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Анотація:

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 the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.

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34

Geurts, Maarten H., Shashank Gandhi, Matteo G. Boretto, Ninouk Akkerman, Lucca L. M. Derks, Gijs van Son, Martina Celotti, et al. "One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids." Nature Communications 14, no. 1 (August 17, 2023). http://dx.doi.org/10.1038/s41467-023-40701-3.

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AbstractOptimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

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35

Marquart, Kim F., Ahmed Allam, Sharan Janjuha, Anna Sintsova, Lukas Villiger, Nina Frey, Michael Krauthammer, and Gerald Schwank. "Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens." Nature Communications 12, no. 1 (August 25, 2021). http://dx.doi.org/10.1038/s41467-021-25375-z.

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Анотація:

AbstractBase editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable transition of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have great potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.

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36

Xue, Niannian, Xu Liu, Dan Zhang, Youming Wu, Yi Zhong, Jinxin Wang, Wenjing Fan, et al. "Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD." Nature Communications 14, no. 1 (March 3, 2023). http://dx.doi.org/10.1038/s41467-023-36887-1.

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Анотація:

AbstractBase editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and the A/C simultaneous conversion of the dual base editor hinder their broad applications. In this study, through fusion of ABE8e with Rad51 DNA-binding domain, we generate a hyperactive ABE (hyABE) which offers improved A-to-G editing efficiency at the region (A10-A15) proximal to the PAM, with 1.2- to 7-fold improvement compared to ABE8e. Similarly, we develop optimized dual base editors (eA&C-BEmax and hyA&C-BEmax) with markedly improved simultaneous A/C conversion efficiency (1.2-fold and 1.5-fold improvement, respectively) compared to A&C-BEmax in human cells. Moreover, these optimized base editors catalyze efficiently nucleotide conversions in zebrafish embryos to mirror human syndrome or in human cells to potentially treat genetic diseases, indicating their great potential in broad applications for disease modeling and gene therapy.

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37

Fan, Jiao, Yige Ding, Chao Ren, Ziguo Song, Jie Yuan, Qiuzhen Chen, Chenchen Du, Chao Li, Xiaolong Wang, and Wenjie Shu. "Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing." Communications Biology 4, no. 1 (July 16, 2021). http://dx.doi.org/10.1038/s42003-021-02406-5.

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AbstractCytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

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38

Lee, Hye Kyung, Harold E. Smith, Chengyu Liu, Michaela Willi, and Lothar Hennighausen. "Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos." Communications Biology 3, no. 1 (January 9, 2020). http://dx.doi.org/10.1038/s42003-019-0745-3.

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Анотація:

AbstractDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

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39

Chen, Qi, Yangning Sun, Jia Yao, Yingfan Lu, Ruikang Qiu, Fuling Zhou, Zixin Deng, and Yuhui Sun. "Engineering of Peptide‐Inserted Base Editors with Enhanced Accuracy and Security." Small, February 25, 2025. https://doi.org/10.1002/smll.202411583.

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Анотація:

AbstractBase editors are effective tools for introducing base conversions without double‐strand breaks, showing broad applications in biotechnological and clinical areas. However, their non‐negligible bystander mutations and off‐target effects have raised extensive safety concerns. To address these issues, a novel method is developed by inserting specific peptide fragments into the substrate binding pocket of deaminases in base editors to modify these outcomes. It is validated that the composition and position of the inserted peptide can significantly impact the performance of A3A‐based cytosine base editor and TadA‐8e‐based adenine base editor, leading to improved editing activity and precision in human HEK293T cells. Importantly, the TadA‐8e variant with DPLVLRRRQ peptide inserted behind S116 residue showed a strong motif preference of Y4A5N6, which can accurately edit the A5 base in targeted protospacer with minimized bystander and off‐target effects in DNA and RNA‐level. By summarizing the regularity during engineering, a set of systematic procedures is established, which can potentially be used to modify other types of base editors and make them more accurate and secure. In addition, the peptide insertion strategy is also proven to be compatible with traditional amino acid changes which have been reported, exhibiting excellent compatibility.

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40

McGrath, Erica, Hyunsu Shin, Linyi Zhang, Je-Nie Phue, Wells W. Wu, Rong-Fong Shen, Yoon-Young Jang, Javier Revollo, and Zhaohui Ye. "Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing." Nature Communications 10, no. 1 (November 25, 2019). http://dx.doi.org/10.1038/s41467-019-13342-8.

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Анотація:

AbstractDNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.

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Neugebauer, Monica E., Alvin Hsu, Mandana Arbab, Nicholas A. Krasnow, Amber N. McElroy, Smriti Pandey, Jordan L. Doman, et al. "Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity." Nature Biotechnology, November 10, 2022. http://dx.doi.org/10.1038/s41587-022-01533-6.

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AbstractCytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

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42

Fan, Tingting, Yanhao Cheng, Yuechao Wu, Shishi Liu, Xu Tang, Yao He, Shanyue Liao, et al. "High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants." Nature Communications 15, no. 1 (June 14, 2024). http://dx.doi.org/10.1038/s41467-024-49473-w.

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Анотація:

AbstractCytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox.

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Hao, Wenliang, Wenjing Cui, Zhongyi Cheng, Laichuang Han, Feiya Suo, Zhongmei Liu, Li Zhou, and Zhemin Zhou. "Development of a base editor for protein evolution via in situ mutation in vivo." Nucleic Acids Research, August 14, 2021. http://dx.doi.org/10.1093/nar/gkab673.

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Анотація:

Abstract Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.

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Yang, Chao, Zhenzhen Ma, Keshan Wang, Xingxiao Dong, Meiyu Huang, Yaqiu Li, Xiagu Zhu, et al. "HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing." Nature Communications 14, no. 1 (April 27, 2023). http://dx.doi.org/10.1038/s41467-023-38193-2.

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AbstractC-to-G base editors have been successfully constructed recently, but limited work has been done on concurrent C-to-G and A-to-G base editing. In addition, there is also limited data on how chromatin-associated factors affect the base editing. Here, we test a series of chromatin-associated factors, and chromosomal protein HMGN1 was found to enhance the efficiency of both C-to-G and A-to-G base editing. By fusing HMGN1, GBE and ABE to Cas9, we develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE) which is capable of converting simultaneous A and C to G conversion with substantial editing efficiency. Accordingly, the HMGN1 role shown in this work and the resulting GGBE tool further broaden the genome manipulation capacity of CRISPR-directed base editors.

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45

Zhao, Yu, Dantong Shang, Ruhong Ying, Hanhua Cheng, and Rongjia Zhou. "An optimized base editor with efficient C-to-T base editing in zebrafish." BMC Biology 18, no. 1 (December 2020). http://dx.doi.org/10.1186/s12915-020-00923-z.

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Abstract Background Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. Results Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). Conclusions Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

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46

Luo, Juan, Muhammad Abid, Jing Tu, Xinxia Cai, Yi Zhang, Puxin Gao, and Hongwen Huang. "Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana." BMC Plant Biology 23, no. 1 (June 7, 2023). http://dx.doi.org/10.1186/s12870-023-04322-8.

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Анотація:

Abstract Background The base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration. Results In the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs containing A3A, A3A (Y130F), and rAPOBEC1(R33A) to compare their base editing efficiency in allotetraploid N. benthamiana (n = 4x). We used 14 target sites to compare their editing efficiency using transient transformation in tobacco plants. The sanger sequencing and deep sequencing results showed that A3A-CBE was the most efficient base editor. In addition, the results showed that A3A-CBE provided most comprehensive editing window (C1 ~ C17 could be edited) and had a better editing efficiency under the base background of TC. The target sites (T2 and T6) analysis in transformed N. benthamiana showed that only A3A-CBE can have C-to-T editing events and the editing efficiency of T2 was higher than T6. Additionally, no off-target events were found in transformed N. benthamiana. Conclusions All in all, we conclude that A3A-CBE is the most suitable vector for specific C to T conversion in N. benthamiana. Current findings will provide valuable insights into selecting an appropriate base editor for breeding polyploid plants.

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Dickson, Kristie-Ann, Natisha Field, Tiane Blackman, Yue Ma, Tao Xie, Ecem Kurangil, Sobia Idrees, et al. "CRISPR single base-editing: in silico predictions to variant clonal cell lines." Human Molecular Genetics, June 27, 2023. http://dx.doi.org/10.1093/hmg/ddad105.

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Abstract Engineering single base edits using CRISPR technology including specific deaminases and single-guide RNA (sgRNA) is a rapidly evolving field. Different types of base edits can be constructed, with cytidine base editors (CBEs) facilitating transition of C-to-T variants, adenine base editors (ABEs) enabling transition of A-to-G variants, C•G-to-G•C transversion base editors (CGBEs) and recently adenine transversion editors (AYBE) that create A-to-C and A-to-T variants. The base-editing machine learning algorithm BE-Hive predicts which sgRNAs and base editor constructs have the strongest likelihood of achieving desired base edits. We have used BE-Hive and TP53 mutation data from The Cancer Genome Atlas ovarian cancer cohort to predict which mutations can be engineered, or reverted to wild-type sequence, using CBEs, ABEs or CGBEs. We have developed and automated a ranking system to assist in selecting optimally designed sgRNA that considers the presence of a suitable protospacer adjacent motif (PAM), the frequency of predicted bystander edits, editing efficiency and target base change. We have generated single constructs containing ABE or CBE editing machinery, an sgRNA cloning backbone and an enhanced green fluorescent protein tag (EGFP), removing the need for co-transfection of multiple plasmids. We have tested our ranking system and new plasmid constructs to engineer the p53 mutants Y220C, R282W and R248Q into wild-type p53 cells and shown that these mutants cannot activate four p53 target genes, mimicking the behaviour of endogenous p53 mutations. This field will continue to rapidly progress, requiring new strategies such as we propose to ensure desired base-editing outcomes.

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48

Wang, Dong, Yani Chen, Tao Zhu, Jie Wang, Man Liu, Shujuan Tian, Jiafa Wang, and Li Yuan. "Developing a highly efficient CGBE base editor in watermelon." Horticulture Research, August 1, 2023. http://dx.doi.org/10.1093/hr/uhad155.

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Abstract Cytosine and adenosine base editors (CBEs and ABEs) are novel genome-editing tools that have been widely utilized in molecular breeding to precisely modify single-nucleotide polymorphisms (SNPs) critical for plant agronomic traits and species evolution. However, conventional BE editors are limited to achieve C-to-T and A-to-G substitutions, respectively. To enhance the applicability of base editing technology in watermelon, we developed an efficient CGBE editor (SCGBE2.0) by removing the UGI unit from the commonly used hA3A-CBE and incorporating the UNG component. Seven specific guide RNAs (sgRNAs) targeting five watermelon genes were designed to assess the editing efficiency of SCGBE. The results obtained from stably transformed watermelon plants demonstrated that SCGBE2.0 could efficiently induce C-to-G mutations at positions C5-C9 in 43.2% transgenic plants (with a maximum base conversion efficiency of 46.1%) and C-to-A mutation at position C4 in 23.5% transgenic plants (with a maximum base conversion efficiency of 45.9%). These findings highlight the capability of our integrated SCGBE2.0 editor to achieve C-to-G/A mutations in a site-preferred manner, thus providing an efficient base editing tool for precise base modification and site-directed saturated mutagenesis in watermelon.

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49

Sretenovic, Simon, Shishi Liu, Gen Li, Yanhao Cheng, Tingting Fan, Yang Xu, Jianping Zhou, et al. "Exploring C-To-G Base Editing in Rice, Tomato, and Poplar." Frontiers in Genome Editing 3 (September 15, 2021). http://dx.doi.org/10.3389/fgeed.2021.756766.

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As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9’s canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

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50

"Base editors." Nature Biotechnology 39, no. 8 (August 2021): 917. http://dx.doi.org/10.1038/s41587-021-01015-1.

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