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.

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.

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 Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

2021 ◽  
Author(s):  
Han Zhang ◽  
Nathan Bamidele ◽  
Pengpeng Liu ◽  
Ogooluwa Ojelabi ◽  
Xin D. Gao ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Cell Types ◽  
Vector System ◽  
Adeno Associated Virus ◽  
Base Editing ◽  
Guide Rna ◽  
Gene Therapies ◽  
Compact Size ◽  
Adenine Base

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

scholarly journals In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

2021 ◽  
Author(s):  
Tanja Rothgangl ◽  
Melissa K. Dennis ◽  
Paulo J. C. Lin ◽  
Rurika Oka ◽  
Dominik Witzigmann ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Point Mutations ◽  
Density Lipoprotein ◽  
Negative Regulator ◽  
Base Editing ◽  
Guide Rna ◽  
Humoral Immune ◽  
Cholesterol Levels ◽  
Adenine Base

AbstractMost known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

scholarly journals Cytosine but not adenine base editor generates mutations in mice

2019 ◽  
Author(s):  
Hye Kyung Lee ◽  
Harold E. Smith ◽  
Chengyu Liu ◽  
Michaela Willi ◽  
Lothar Hennighausen
Keyword(s):  
Point Mutations ◽  
Living Cells ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
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 and its ultimate success therapeutically depends on its accuracy. Here we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editor (ABE) in mouse embryos using unbiased whole genome sequencing of a family-based trio cohort. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls.

scholarly journals Base editing generates substantial off-target single nucleotide variants

2018 ◽  
Author(s):  
Erwei Zuo ◽  
Yidi Sun ◽  
Wu Wei ◽  
Tanglong Yuan ◽  
Wenqin Ying ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biomedical Application ◽  
Spontaneous Mutation ◽  
Great Promise ◽  
Single Nucleotide Variants ◽  
Spontaneous Mutation Rate ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Target Effects

AbstractGenome editing tools including CRISPR/Cas9 and base editors hold great promise for correcting pathogenic mutations. Unbiased genome-wide off-target effects of the editing in mammalian cells is required before clinical applications, but determination of the extent of off-target effects has been difficult due to the existence of single nucleotide polymorphisms (SNPs) in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations without interference of SNPs. We applied GOTI to both the CRISPR-Cas9 and base editing (BE3) systems by editing one blastomere of the two-cell mouse embryo and then compared whole genome sequences of progeny-cell populations at E14.5 stage. Sequence analysis of edited and non-edited cell progenies showed that undesired off-target single nucleotide variants (SNVs) are rare (average 10.5) in CRISPR-edited mouse embryos, with a frequency close to the spontaneous mutation rate. By contrast, BE3 editing induced over 20-fold higher SNVs (average 283), raising the concern of using base-editing approaches for biomedical application.

scholarly journals An optimized toolkit for precision base editing

2018 ◽  
Author(s):  
Maria Paz Zafra ◽  
Emma M Schatoff ◽  
Alyna Katti ◽  
Miguel Foronda ◽  
Marco Breinig ◽  
...  
Keyword(s):  
Protein Sequences ◽  
Model Systems ◽  
Genetic Mutations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
Vector Systems ◽  
Wide Range ◽  
Single Base Pair

AbstractCRISPR base editing is a potentially powerful technology that enables the creation of genetic mutations with single base pair resolution. By re-engineering both DNA and protein sequences, we developed a collection of constitutive and inducible base editing vector systems that dramatically improve the ease and efficiency by which single nucleotide variants can be created. This new toolkit is effective in a wide range of model systems, and provides a means for efficientin vivosomatic base editing.

scholarly journals Combination of Genome-Wide Polymorphisms and Copy Number Variations of Pharmacogenes in Koreans

2021 ◽  
Vol 11 (1) ◽  
pp. 33
Author(s):  
Nayoung Han ◽  
Jung Mi Oh ◽  
In-Wha Kim
Keyword(s):  
Copy Number ◽  
Genome Wide Association Study ◽  
Copy Number Gain ◽  
Copy Number Variations ◽  
Gene Gain ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Haplotype Blocks ◽  
Genome Wide ◽  
Control And Prevention

For predicting phenotypes and executing precision medicine, combination analysis of single nucleotide variants (SNVs) genotyping with copy number variations (CNVs) is required. The aim of this study was to discover SNVs or common copy CNVs and examine the combined frequencies of SNVs and CNVs in pharmacogenes using the Korean genome and epidemiology study (KoGES), a consortium project. The genotypes (N = 72,299) and CNV data (N = 1000) were provided by the Korean National Institute of Health, Korea Centers for Disease Control and Prevention. The allele frequencies of SNVs, CNVs, and combined SNVs with CNVs were calculated and haplotype analysis was performed. CYP2D6 rs1065852 (c.100C>T, p.P34S) was the most common variant allele (48.23%). A total of 8454 haplotype blocks in 18 pharmacogenes were estimated. DMD ranked the highest in frequency for gene gain (64.52%), while TPMT ranked the highest in frequency for gene loss (51.80%). Copy number gain of CYP4F2 was observed in 22 subjects; 13 of those subjects were carriers with CYP4F2*3 gain. In the case of TPMT, approximately one-half of the participants (N = 308) had loss of the TPMT*1*1 diplotype. The frequencies of SNVs and CNVs in pharmacogenes were determined using the Korean cohort-based genome-wide association study.

scholarly journals Prediction of genome-wide effects of single nucleotide variants on transcription factor binding

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sebastian Carrasco Pro ◽  
Katia Bulekova ◽  
Brian Gregor ◽  
Adam Labadorf ◽  
Juan Ignacio Fuxman Bass
Keyword(s):  
Binding Sites ◽  
Cancer Type ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Regulatory Regions ◽  
Genome Wide ◽  
Transcriptional Regulatory ◽  
Gene Regulatory ◽  
The Impact ◽  
The Relationship

Abstract Single nucleotide variants (SNVs) located in transcriptional regulatory regions can result in gene expression changes that lead to adaptive or detrimental phenotypic outcomes. Here, we predict gain or loss of binding sites for 741 transcription factors (TFs) across the human genome. We calculated ‘gainability’ and ‘disruptability’ scores for each TF that represent the likelihood of binding sites being created or disrupted, respectively. We found that functional cis-eQTL SNVs are more likely to alter TF binding sites than rare SNVs in the human population. In addition, we show that cancer somatic mutations have different effects on TF binding sites from different TF families on a cancer-type basis. Finally, we discuss the relationship between these results and cancer mutational signatures. Altogether, we provide a blueprint to study the impact of SNVs derived from genetic variation or disease association on TF binding to gene regulatory regions.

scholarly journals Impact of Pre and Post Variant Filtration Strategies on Imputation

2020 ◽  
Author(s):  
Celine Charon ◽  
Rodrigue Allodji ◽  
Vincent Meyer ◽  
Jean-François Deleuze
Keyword(s):  
Quality Control ◽  
Rare Variants ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Nucleotide Polymorphisms ◽  
Direct Effects ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Conservative Post

Abstract Quality control methods for genome-wide association studies and fine mapping are commonly used for imputation, however, they result in loss of many single nucleotide polymorphisms (SNPs). To investigate the consequences of filtration on imputation, we studied the direct effects on the number of markers, their allele frequencies, imputation quality scores and post-filtration events. We pre-phrased 1,031 genotyped individuals from diverse ethnicities and compared the imputed variants to 1,089 NCBI recorded individuals for additional validation.Without variant pre-filtration based on quality control (QC), we observed no impairment in the imputation of SNPs that failed QC whereas with pre-filtration there was an overall loss of information. Significant differences between frequencies with and without pre-filtration were found only in the range of very rare (5E-04-1E-03) and rare variants (1E-03-5E-03) (p < 1E-04). Increasing the post-filtration imputation quality score from 0.3 to 0.8 reduced the number of single nucleotide variants (SNVs) <0.001 2.5 fold with or without QC pre-filtration and halved the number of very rare variants (5E-04). As a result, to maintain confidence and enough SNVs, we propose here a 2-step post-filtration approach to increase the number of very rare and rare variants compared to conservative post-filtration methods.

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