scholarly journals A general theoretical framework to design base editors with reduced bystander effects

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qian Wang ◽  
Jie Yang ◽  
Zhicheng Zhong ◽  
Jeffrey A. Vanegas ◽  
Xue Gao ◽  
...  
Keyword(s):  
Bystander Effect ◽  
Point Mutations ◽  
Stochastic Approach ◽  
Medical Applications ◽  
Dynamic Simulations ◽  
Bystander Effects ◽  
Discrete State ◽  
Base Editing ◽  
Different Levels ◽  
New Mutations

AbstractBase editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 – 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the “TC” motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.

scholarly journals A General Theoretical Framework to Design Base Editors with Reduced Bystander Effects

2021 ◽  
Author(s):  
Qian Wang ◽  
Jie Yang ◽  
Zhicheng Zhong ◽  
Xue Gao ◽  
Anatoly Kolomeisky
Keyword(s):  
Experimental Data ◽  
Gene Therapy ◽  
High Precision ◽  
Bystander Effect ◽  
Stochastic Approach ◽  
Molecular Dynamic Simulations ◽  
Dynamic Simulations ◽  
Bystander Effects ◽  
Discrete State ◽  
Base Editing

Abstract Base editors (BEs) hold great potential for gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 - 10 nucleotides). To assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that describes the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model well reproduces the experimental data of A3A-BE3 and its variants for target and bystander editing. Building upon this model, we propose several general principles that can guide the design of BEs with a reduced bystander effect. We used these principles to improve the A3G-BEs with high precision and verified their base-editing activities experimentally. In summary, our study provides a computational-aided platform to assist in designing BEs with reduced bystander effects.

scholarly journals One-step multiple site-specific base editing by direct embryo injection for precision and pyramid pig breeding

2020 ◽  
Author(s):  
Ruigao Song ◽  
Yu Wang ◽  
Qiantao Zheng ◽  
Jing Yao ◽  
Chunwei Cao ◽  
...  
Keyword(s):  
Stop Codon ◽  
Point Mutations ◽  
Embryonic Cells ◽  
Site Specific ◽  
Editing Efficiency ◽  
Base Editing ◽  
One Step ◽  
Pig Performance ◽  
Novel Alleles

AbstractPrecise and simultaneous acquisition of multiple beneficial alleles in the genome to improve pig performance are pivotal for making elite breeders. Cytidine base editors (CBEs) have emerged as powerful tools for site-specific single nucleotide replacement. Here, we compare the editing efficiency of four CBEs in porcine embryonic cells and embryos to show that hA3A-BE3-Y130F and hA3A-eBE3-Y130F consistently results in higher base-editing efficiency and lower toxic effects to in vitro embryo development. We also show that zygote microinjection of hA3A-BE3-Y130F results in one-step generation of pigs (3BE pigs) harboring C-to-T point mutations, including a stop codon in CD163 and in MSTN and induce beneficial allele in IGF2. The 3BE pigs showed improved growth performance, hip circumference, food conversion rate. Our results demonstrate that CBEs can mediate high throughput genome editing by direct embryo microinjection. Our approach allows immediate introduction of novel alleles for beneficial traits in transgene-free animals for pyramid breeding.

scholarly journals ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Jingke Xie ◽  
Xingyun Huang ◽  
Xia Wang ◽  
Shixue Gou ◽  
Yanhui Liang ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Multiple Point ◽  
Nucleotide Polymorphisms ◽  
Spacer Length ◽  
Endogenous Gene ◽  
Base Editing ◽  
Fetal Fibroblasts

Abstract Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

scholarly journals Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hye Kyung Lee ◽  
Harold E. Smith ◽  
Chengyu Liu ◽  
Michaela Willi ◽  
Lothar Hennighausen
Keyword(s):  
Point Mutations ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Wide Range ◽  
Family Based ◽  
Correct Point ◽  
Adenine Base

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.

Monozygotic Twins with Apert Syndrome

2008 ◽  
Vol 45 (1) ◽  
pp. 101-104 ◽  
Author(s):  
Corstiaan C. Breugem ◽  
Donald F. Fitzpatrick ◽  
Cynthia Verchere
Keyword(s):  
English Literature ◽  
Point Mutations ◽  
Monozygotic Twins ◽  
Growth Factor Receptor ◽  
Clinical Findings ◽  
Apert Syndrome ◽  
Normal Children ◽  
Metopic Synostosis ◽  
Metopic Suture ◽  
New Mutations

Apert syndrome results almost exclusively from one of two point mutations (Ser252Trp or Pro253Arg) in fibroblast growth factor receptor 2. Most patients with Apert syndrome have this as an autosomal dominant abnormality. The majority of cases are sporadic, resulting from new mutations. Although there have been some descriptions of familial Apert syndrome, we could find only one previous description in the English literature about twinning in Apert syndrome. This report demonstrates monozygotic twins affected by Apert syndrome with both boys having the Ser252Trp mutation. Although the general constellation of clinical findings was characteristic for Apert syndrome, this case report is unique since the twins had different craniofacial and hand features. One of our twins had a metopic synostosis while Apert syndrome is often characterized by the large metopic suture that closes much later when compared to normal children.

scholarly journals Exosomal MiR-769-5p Exacerbates Ultraviolet-Induced Bystander Effect by Targeting TGFBR1

2020 ◽  
Vol 11 ◽  
Author(s):  
Na Ni ◽  
Weiwei Ma ◽  
Yanling Tao ◽  
Juan Liu ◽  
Hui Hua ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Human Skin ◽  
Bystander Effect ◽  
Transforming Growth Factor ◽  
Underlying Mechanism ◽  
Skin Fibroblasts ◽  
Luciferase Reporter ◽  
Human Skin Fibroblasts ◽  
Bystander Effects ◽  
Damage Level

Exosomal microRNAs have been investigated in bystander effect, but it is unclear whether microRNA works in ultraviolet radiation–induced bystander effects (UV-RIBEs) and what the underlying mechanism could be. Exosomes from ultraviolet (UV)–irradiated human skin fibroblasts (HSFs) were isolated and transferred to normal HSFs, followed by the detection of proliferation rate, oxidative damage level, and apoptosis rate. Exosomal miRNAs were evaluated and screened with miRNA sequencing and quantitative reverse transcriptase–polymerase chain reaction method. MiRNA shuttle and bystander photodamage reactions were observed after transfection of miR-769-5p. MiR-769-5p targeting gene transforming growth factor-β1 (TGFBR1), and TGFBR1 mRNA 3′-untranslated region (UTR) was assessed and identified by Western blotting and dual-luciferase reporter assay. Bystander effects were induced after being treated with isolated exosomes from UV-irradiated HSFs. Exosomal miR-769-5p expression was significantly upregulated. Human skin fibroblasts showed lower proliferation, increasing oxidative damage, and faster occurrence of apoptosis after transfection. Exosome-mediated transfer of miR-769-5p was observed. Upregulation of miR-769-5p induced bystander effects, whereas downregulation of miR-769-5p can suppress UV-RIBEs. In addition, miR-769-5p was found to downregulate TGFBR1 gene expression by directly targeting its 3′-UTR. Our results demonstrate that exosome-mediated miR-769-5p transfer could function as an intercellular messenger and exacerbate UV-RIBEs. MiR-769-5p inhibits the expression of TGFBR1 by targeting TGFBR1 mRNA 3′-UTR.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

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

scholarly journals Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics

Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 55
Author(s):  
Linda Celeste Montemiglio ◽  
Elena Gugole ◽  
Ida Freda ◽  
Cécile Exertier ◽  
Lucia D’Auria ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Substrate Binding ◽  
Point Mutations ◽  
Conformational Space ◽  
Amino Acid Side Chain ◽  
Dynamic Simulations ◽  
Substrate Protein ◽  
Contact Points ◽  
Solvent Molecules

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

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