High-fidelity base editor with no detectable genome-wide off-target effects

2020 ◽  
Author(s):  
Erwei Zuo ◽  
Yidi Sun ◽  
Tanglong Yuan ◽  
Bingbing He ◽  
Changyang Zhou ◽  
...  
Keyword(s):  
Rna Binding ◽  
High Fidelity ◽  
Sequencing Analysis ◽  
Single Nucleotide ◽  
Dna And Rna ◽  
Genome Wide ◽  
Cell Embryo ◽  
Single Nucleotide Variations ◽  
Target Rna ◽  
Target Effects

Base editors hold promise for correcting pathogenic mutations, while substantial single nucleotide variations (SNVs) on both DNA and RNA were generated by cytosine base editors (CBEs). Here we examined possibilities to reduce off-target effects by engineering cytosine deaminases. By screening 24 CBEs harboring various rAPOBEC1 (BE3) or human APOBEC3A (BE3-hA3A) mutations on the ssDNA or RNA binding domain, we found 8 CBE variations could maintain high on-target editing efficiency. Using Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) method and RNA sequencing analysis, we found DNA off-target SNVs induced by BE3 could be completely eliminated in BE3R126E but the off-target RNA SNVs was only slightly reduced. By contrast, BE3-hA3AY130F abolished the RNA off-target effects while could not reduce the DNA off-target effects. Notably, BE3R132E, BE3W90Y+R126E and BE3W90F+R126E achieved the elimination of off-target SNVs on both DNA and RNA, suggesting the feasibility of engineering base editors for high fidelity deaminases.

scholarly journals Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

Science ◽  
2019 ◽  
pp. eaav9973 ◽  
Author(s):  
Erwei Zuo ◽  
Yidi Sun ◽  
Wu Wei ◽  
Tanglong Yuan ◽  
Wenqin Ying ◽  
...  
Keyword(s):  
Spontaneous Mutation ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Spontaneous Mutation Rate ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Cell Embryo ◽  
Adenine Base ◽  
Target Effects

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

scholarly journals Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects

2020 ◽  
Vol 6 (29) ◽  
pp. eaba1773 ◽  
Author(s):  
Sangsin Lee ◽  
Ning Ding ◽  
Yidi Sun ◽  
Tanglong Yuan ◽  
Jing Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Precision ◽  
Disease Modeling ◽  
Human Cells ◽  
Sequencing Analysis ◽  
Injection Method ◽  
Cell Embryo ◽  
Human Apobec3g ◽  
Target Effects

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

scholarly journals Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jiao Fan ◽  
Yige Ding ◽  
Chao Ren ◽  
Ziguo Song ◽  
Jie Yuan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Double Strand Breaks ◽  
Great Promise ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Dna Base ◽  
Target Dna ◽  
Single Nucleotide Variations ◽  
Adenine Deaminase ◽  
Target Rna

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.

scholarly journals Clonal Lineages of Erysipelothrix rhusiopathiae Responsible for Acute Swine Erysipelas in Japan Identified by Using Genome-Wide Single-Nucleotide Polymorphism Analysis

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Yohsuke Ogawa ◽  
Kazumasa Shiraiwa ◽  
Yoshitoshi Ogura ◽  
Tadasuke Ooka ◽  
Sayaka Nishikawa ◽  
...  
Keyword(s):  
Polymorphism Analysis ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Erysipelothrix Rhusiopathiae ◽  
Single Nucleotide ◽  
Content Type ◽  
Genome Wide ◽  
Wide Range ◽  
Swine Erysipelas ◽  
Lineage Iv

ABSTRACTErysipelothrix rhusiopathiaecauses swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due toE. rhusiopathiaeserovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34E. rhusiopathiaeserovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene (spaA) revealed that the strains isolated after 2007 exhibited the samespaAgenotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specificspaAgenotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions.IMPORTANCEUsing large-scale whole-genome sequence data fromErysipelothrix rhusiopathiaeisolates from a wide range of hosts and geographic origins, a recent study clarified the existence of three distinct clades (clades 1, 2, and 3) that are found across multiple continents and host species, representing both livestock and wildlife, and an “intermediate” clade between clade 2 and the dominant clade 3 within the species. In this study, we found that theE. rhusiopathiaeJapanese strains examined exhibited remarkably low levels of genetic diversity and confirmed that all of the Japanese and Chinese swine isolates examined in this study belong to clonal lineages within the intermediate clade. We report thatspaAgenotyping ofE. rhusiopathiaestrains is a practical alternative to whole-genome sequencing analysis of theE. rhusiopathiaeisolates from eastern Asian countries.

scholarly journals Genotyping common, large structural variations in 5,202 genomes using pangenomes, the Giraffe mapper, and the vg toolkit

2020 ◽  
Author(s):  
Jouni Sirén ◽  
Jean Monlong ◽  
Xian Chang ◽  
Adam M. Novak ◽  
Jordan M. Eizenga ◽  
...  
Keyword(s):  
Human Populations ◽  
Structural Variations ◽  
Single Nucleotide ◽  
Human Genomes ◽  
Genome Wide ◽  
Sequence Graph ◽  
Long Read ◽  
Comparable Accuracy ◽  
Single Nucleotide Variations ◽  
Allelic Variations

ABSTRACTWe introduce Giraffe, a pangenome short read mapper that can efficiently map to a collection of haplotypes threaded through a sequence graph. Giraffe, part of the variation graph toolkit (vg)1, maps reads to thousands of human genomes at around the same speed BWA-MEM2 maps reads to a single reference genome, while maintaining comparable accuracy to VG-MAP, vg’s original mapper. We have developed efficient genotyping pipelines using Giraffe. We demonstrate improvements in genotyping for single nucleotide variations (SNVs), insertions and deletions (indels) and structural variations (SVs) genome-wide. We use Giraffe to genotype and phase 167 thousands structural variations ascertained from long read studies in 5,202 human genomes sequenced with short reads, including the complete 1000 Genomes Project dataset, at an average cost of $1.50 per sample. We determine the frequency of these variations in diverse human populations, characterize their complex allelic variations and identify thousands of expression quantitative trait loci (eQTLs) driven by these variations.

scholarly journals Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map

2020 ◽  
Author(s):  
Alli L. Gombolay ◽  
Francesca Storici
Keyword(s):  
Computing Time ◽  
Wide Distribution ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Hands On ◽  
Nucleotide Resolution ◽  
User Friendly ◽  
Single Nucleotide Resolution

ABSTRACTRibose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze raw ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides, and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 hr of computing time for most datasets and about 30 min of hands-on time.

Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽  
2019 ◽  
pp. eaaw7166 ◽  
Author(s):  
Shuai Jin ◽  
Yuan Zong ◽  
Qiang Gao ◽  
Zixu Zhu ◽  
Yanpeng Wang ◽  
...  
Keyword(s):  
High Fidelity ◽  
Single Nucleotide Variants ◽  
Disease Treatment ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Wide ◽  
Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification

2005 ◽  
Vol 26 (3) ◽  
pp. 710-715 ◽  
Author(s):  
Mladen V. Tzvetkov ◽  
Christian Becker ◽  
Bettina Kulle ◽  
Peter Nürnberg ◽  
Jürgen Brockmöller ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Whole Genome Amplification ◽  
High Fidelity ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Genome Amplification ◽  
Genome Wide ◽  
Displacement Based

scholarly journals Genome-wide identifying of G-quadruplex structures directly by whole-genome resequencing

2021 ◽  
Author(s):  
Jing Tu ◽  
Mengqin Duan ◽  
Wenli Liu ◽  
Na Lu ◽  
Xiao Sun ◽  
...  
Keyword(s):  
Individual Differences ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Single Nucleotide ◽  
Genome Wide ◽  
G Quadruplex ◽  
Sequencing Quality ◽  
Single Nucleotide Variations ◽  
Whole Genome Resequencing ◽  
The Given

Abstract We present a convenient genome-wide DNA G-quadruplex (G4) profiling method that identifies G4 structures from ordinary whole-genome resequencing data by seizing the slight fluctuation of sequencing quality. We identified 736,689 G4 structures within human genome, in which 44.9% of all predicted canonical G4-froming sequences were contained. We observed that some of the single nucleotide variations (SNVs) influenced the formation of G4 structures, including homozygous SNVs and heterozygous SNVs. Due to SNVs contain individual differences, the given approach is available to identify and characterize G4s genome-wide for specific individuals.

scholarly journals High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects

Nature ◽  
2016 ◽  
Vol 529 (7587) ◽  
pp. 490-495 ◽  
Author(s):  
Benjamin P. Kleinstiver ◽  
Vikram Pattanayak ◽  
Michelle S. Prew ◽  
Shengdar Q. Tsai ◽  
Nhu T. Nguyen ◽  
...  
Keyword(s):  
High Fidelity ◽  
Genome Wide ◽  
Target Effects
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