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Author: Grafiati
Published: 14 March 2023

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1

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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2

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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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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4

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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5

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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6

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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7

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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8

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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9

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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10

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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11

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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12

Li, Chang, Aphrodite Georgakopoulou, Arpit Mishra, Sucheol Gil, R. David Hawkins, Evangelia Yannaki, and 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, no. 4 (February 23, 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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13

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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14

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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15

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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16

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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17

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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18

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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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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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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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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23

Molla, Kutubuddin A., Simon Sretenovic, Kailash C. Bansal, and Yiping Qi. "Precise plant genome editing using base editors and prime editors." Nature Plants 7, no. 9 (September 2021): 1166–87. http://dx.doi.org/10.1038/s41477-021-00991-1.

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24

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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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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Hwang, Gue-Ho, Beomjong Song, and Sangsu Bae. "Current widely-used web-based tools for CRISPR nucleases, base editors, and prime editors." Gene and Genome Editing 1 (June 2021): 100004. http://dx.doi.org/10.1016/j.ggedit.2021.100004.

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26

Yang, Bei, Li Yang, and Jia Chen. "Development and Application of Base Editors." CRISPR Journal 2, no. 2 (April 2019): 91–104. http://dx.doi.org/10.1089/crispr.2019.0001.

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27

Wrighton, Katharine H. "Cytosine base editors go off-target." Nature Reviews Genetics 20, no. 5 (March 13, 2019): 254–55. http://dx.doi.org/10.1038/s41576-019-0110-x.

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Zeng, Dongchang, Zhiye Zheng, Yuxin Liu, Taoli Liu, Tie Li, Jianhong Liu, Qiyu Luo, et al. "Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools." International Journal of Molecular Sciences 23, no. 14 (July 20, 2022): 7990. http://dx.doi.org/10.3390/ijms23147990.

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CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.
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Stafford, Tom, and Deborah J. Armstrong. "From the Editors." ACM SIGMIS Database: the DATABASE for Advances in Information Systems 52, SI (December 9, 2021): 5–6. http://dx.doi.org/10.1145/3505639.3505641.

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Friends, Deb Armstrong and I have reached the culmination of our editorial term, and K.D. Joshi of University of Nevada-Reno has been chosen as the new Editor-in-Chief. Professor Joshi is a long-time contributor, Senior Editor, and thought leader in our parent organization, ACM SIGMIS, and its venerable CPR conference. K.D. has a marvelous vision, superb connections with our ACM SIGMIS CPR constituency, and an operational record second to none. She will be a great leader for continuing the growth trajectory of The DATA BASE for Advances in Information Systems.
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Standage-Beier, Kylie, Stefan J. Tekel, Nicholas Brookhouser, Grace Schwarz, Toan Nguyen, Xiao Wang, and David A. Brafman. "A transient reporter for editing enrichment (TREE) in human cells." Nucleic Acids Research 47, no. 19 (August 20, 2019): e120-e120. http://dx.doi.org/10.1093/nar/gkz713.

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Abstract Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.
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Jiang, Lurong, Jie Long, Yang Yang, Lifang Zhou, Jing Su, Fengming Qin, Wenling Tang, Rui Tao, Qiang Chen, and Shaohua Yao. "Internally inlaid SaCas9 base editors enable window specific base editing." Theranostics 12, no. 10 (2022): 4767–78. http://dx.doi.org/10.7150/thno.70869.

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Anzalone, Andrew V., Luke W. Koblan, and David R. Liu. "Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors." Nature Biotechnology 38, no. 7 (June 22, 2020): 824–44. http://dx.doi.org/10.1038/s41587-020-0561-9.

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Jang, Hyeon-Ki, Dong Hyun Jo, Seu-Na Lee, Chang Sik Cho, You Kyeong Jeong, Youngri Jung, Jihyeon Yu, Jeong Hun Kim, Jae-Sung Woo, and Sangsu Bae. "High-purity production and precise editing of DNA base editing ribonucleoproteins." Science Advances 7, no. 35 (August 2021): eabg2661. http://dx.doi.org/10.1126/sciadv.abg2661.

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Ribonucleoprotein (RNP) complex–mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector–mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM–targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.
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Zheng, Shuwen, Haiwen Zhong, Xiaoqing Zhou, Min Chen, Wansheng Li, Yin Zi, Yue Chi, et al. "Efficient and Safe Editing of Porcine Endogenous Retrovirus Genomes by Multiple-Site Base-Editing Editor." Cells 11, no. 24 (December 8, 2022): 3975. http://dx.doi.org/10.3390/cells11243975.

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Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors.
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Griffith, Audrey, Annabel Sangree, Priyanka Roy, Ruth Hanna, and John Doench. "Abstract SY05-02: Applications of base editor technology in small molecule: Target validation." Cancer Research 82, no. 12_Supplement (June 15, 2022): SY05–02—SY05–02. http://dx.doi.org/10.1158/1538-7445.am2022-sy05-02.

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Abstract CRISPR screens that focus on the gene as the unit of information - whether using knockout, interference, or activation - have reinvigorated the field of functional genomics. The recent development of Base Editor technology allows nucleotide-level manipulation, enabling fine-scale investigation of endogenous loci. The low upfront costs of generating a pooled base editing library, coupled with the small scale and relative ease of executing such screens, makes the financial barrier to entry for such experiments low while also not requiring deep experience with screening. One application of this technology is screens that aim to identify resistance mutations in putative targets of small molecules, which provides excellent evidence that the phenotype is caused by on-target activity of the small molecule. This has heretofore typically been an extraordinarily laborious and time-consuming experimental path, by either slowly cultivating spontaneous drug resistant mutants or by generating and screening large scale ORF-variant pools. Base editor screens are well-suited to vastly accelerate this process. Using both A>G and C>T base editors, coupled with variants of the SpCas9 enzyme, we have conducted drug resistance screens with small molecules that target the anti-apoptotic proteins MCL1, BCL2L1, and BCL2, as well as the DNA repair regulator PARP1. These screens identified resistance residues that were also observed in clinical samples, illustrating the utility of this approach. Additionally, base editor screens are excellent tools for studying genetic variants, as we will demonstrate with screens identifying loss-of-function mutations in BRCA1, BRCA2, and TP53. We anticipate that the tools and methodologies described here will facilitate the investigation of impactful variants at a finer and deeper resolution for any locus of interest. Citation Format: Audrey Griffith, Annabel Sangree, Priyanka Roy, Ruth Hanna, John Doench. Applications of base editor technology in small molecule: Target validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY05-02.
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36

Winter, Jackson, and Pablo Perez-Pinera. "Directed Evolution of CRISPR-Cas9 Base Editors." Trends in Biotechnology 37, no. 11 (November 2019): 1151–53. http://dx.doi.org/10.1016/j.tibtech.2019.09.005.

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37

Eid, Ayman, Sahar Alshareef, and Magdy M. Mahfouz. "CRISPR base editors: genome editing without double-stranded breaks." Biochemical Journal 475, no. 11 (June 11, 2018): 1955–64. http://dx.doi.org/10.1042/bcj20170793.

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The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.
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38

Christensen, Chloe L., Rhea E. Ashmead, and Francis Y. M. Choy. "Cell and Gene Therapies for Mucopolysaccharidoses: Base Editing and Therapeutic Delivery to the CNS." Diseases 7, no. 3 (June 26, 2019): 47. http://dx.doi.org/10.3390/diseases7030047.

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Although individually uncommon, rare diseases collectively account for a considerable proportion of disease impact worldwide. A group of rare genetic diseases called the mucopolysaccharidoses (MPSs) are characterized by accumulation of partially degraded glycosaminoglycans cellularly. MPS results in varied systemic symptoms and in some forms of the disease, neurodegeneration. Lack of treatment options for MPS with neurological involvement necessitates new avenues of therapeutic investigation. Cell and gene therapies provide putative alternatives and when coupled with genome editing technologies may provide long term or curative treatment. Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing technology and, more recently, advances in genome editing research, have allowed for the addition of base editors to the repertoire of CRISPR-based editing tools. The latest versions of base editors are highly efficient on-targeting deoxyribonucleic acid (DNA) editors. Here, we describe a number of putative guide ribonucleic acid (RNA) designs for precision correction of known causative mutations for 10 of the MPSs. In this review, we discuss advances in base editing technologies and current techniques for delivery of cell and gene therapies to the site of global degeneration in patients with severe neurological forms of MPS, the central nervous system, including ultrasound-mediated blood-brain barrier disruption.
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39

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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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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40

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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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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41

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 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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42

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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43

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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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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44

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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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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45

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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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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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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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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48

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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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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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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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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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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