Cytosine but not adenine base editor generates mutations in mice

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

Communications Biology ◽

10.1038/s42003-019-0745-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 15

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.

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

Science ◽

10.1126/science.aav9973 ◽

2019 ◽

pp. eaav9973 ◽

Cited By ~ 84

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.

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Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

10.1101/2020.01.06.890244 ◽

2020 ◽

Author(s):

Rabinowitz Roy ◽

Abadi Shiran ◽

Almog Shiri ◽

Offen Daniel

Keyword(s):

Genome Editing ◽

Point Mutations ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Computational Tool ◽

Single Nucleotide ◽

Base Editing ◽

A Genome ◽

Wide Range ◽

Reference Protein

ABSTRACTBase editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. 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 given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

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

Science ◽

10.1126/science.aaw7166 ◽

2019 ◽

pp. eaaw7166 ◽

Cited By ~ 77

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.

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An optimized toolkit for precision base editing

10.1101/303131 ◽

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.

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Systematic identification of non-coding somatic single nucleotide variants associated with altered transcription and DNA methylation in adult and pediatric cancers

NAR Cancer ◽

10.1093/narcan/zcab001 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Fengju Chen ◽

Yiqun Zhang ◽

Chad J Creighton

Keyword(s):

Dna Methylation ◽

Brain Tumor ◽

Low Grade ◽

Single Nucleotide Variants ◽

Multiple Cancer ◽

Single Nucleotide ◽

Altered Expression ◽

Wide Range ◽

Mutational Hotspots ◽

Cancer Types

Abstract Whole-genome sequencing combined with transcriptomics can reveal impactful non-coding single nucleotide variants (SNVs) in cancer. Here, we developed an integrative analytical approach that, as a first step, identifies genes altered in expression or DNA methylation in association with nearby somatic SNVs, in contrast to alternative approaches that first identify mutational hotspots. Using genomic datasets from the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium and the Children's Brain Tumor Tissue Consortium (CBTTC), we identified hundreds of genes and associated CpG islands for which the nearby presence of a non-coding somatic SNV recurrently associated with altered expression or DNA methylation, respectively. Genomic regions upstream or downstream of genes, gene introns and gene untranslated regions were all involved. The PCAWG adult cancer cohort yielded different significant SNV-expression associations from the CBTTC pediatric brain tumor cohort. The SNV-expression associations involved a wide range of cancer types and histologies, as well as potential gain or loss of transcription factor binding sites. Notable genes with SNV-associated increased expression include TERT, COPS3, POLE2 and HDAC2—involving multiple cancer types—MYC, BCL2, PIM1 and IGLL5—involving lymphomas—and CYHR1—involving pediatric low-grade gliomas. Non-coding somatic SNVs show a major role in shaping the cancer transcriptome, not limited to mutational hotspots.

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Computational Analysis of Genetic Code Variations Optimized for the Robustness against Point Mutations with Wobble-like Effects

Life ◽

10.3390/life11121338 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1338

Author(s):

Elena Fimmel ◽

Markus Gumbel ◽

Martin Starman ◽

Lutz Strüngmann

Keyword(s):

Genetic Code ◽

Computational Analysis ◽

Point Mutations ◽

Weighted Graph ◽

Single Nucleotide Variants ◽

Negative Effects ◽

Standard Genetic Code ◽

Single Nucleotide ◽

Random Code ◽

Optimal Weights

It is believed that the codon–amino acid assignments of the standard genetic code (SGC) help to minimize the negative effects caused by point mutations. All possible point mutations of the genetic code can be represented as a weighted graph with weights that correspond to the probabilities of these mutations. The robustness of a code against point mutations can be described then by means of the so-called conductance measure. This paper quantifies the wobble effect, which was investigated previously by applying the weighted graph approach, and seeks optimal weights using an evolutionary optimization algorithm to maximize the code’s robustness. One result of our study is that the robustness of the genetic code is least influenced by mutations in the third position—like with the wobble effect. Moreover, the results clearly demonstrate that point mutations in the first, and even more importantly, in the second base of a codon have a very large influence on the robustness of the genetic code. These results were compared to single nucleotide variants (SNV) in coding sequences which support our findings. Additionally, it was analyzed which structure of a genetic code evolves from random code tables when the robustness is maximized. Our calculations show that the resulting code tables are very close to the standard genetic code. In conclusion, the results illustrate that the robustness against point mutations seems to be an important factor in the evolution of the standard genetic code.

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Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients

Journal of Personalized Medicine ◽

10.3390/jpm10030137 ◽

2020 ◽

Vol 10 (3) ◽

pp. 137

Author(s):

Adrián Montaño ◽

Jesús Hernández-Sánchez ◽

Maribel Forero-Castro ◽

María Matorra-Miguel ◽

Eva Lumbreras ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Wide Spectrum ◽

Lymphoblastic Leukemia ◽

Point Mutations ◽

Genetic Alterations ◽

Copy Number Variations ◽

Single Step ◽

Fusion Genes ◽

Single Nucleotide Variants ◽

Single Nucleotide

Background: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. Methods: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. Results: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. Conclusions: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management.

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Next-generation forward genetic screens: using simulated data to improve the design of mapping-by-sequencing experiments in Arabidopsis

Nucleic Acids Research ◽

10.1093/nar/gkz806 ◽

2019 ◽

Vol 47 (21) ◽

pp. e140-e140

Author(s):

David Wilson-Sánchez ◽

Samuel Daniel Lup ◽

Raquel Sarmiento-Mañús ◽

María Rosa Ponce ◽

José Luis Micol

Keyword(s):

Point Mutations ◽

Sequencing Depth ◽

Nucleotide Polymorphisms ◽

Next Generation ◽

Genetic Screens ◽

Induced Mutations ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Forward Genetic Screens ◽

Mapping By Sequencing

Abstract Forward genetic screens have successfully identified many genes and continue to be powerful tools for dissecting biological processes in Arabidopsis and other model species. Next-generation sequencing technologies have revolutionized the time-consuming process of identifying the mutations that cause a phenotype of interest. However, due to the cost of such mapping-by-sequencing experiments, special attention should be paid to experimental design and technical decisions so that the read data allows to map the desired mutation. Here, we simulated different mapping-by-sequencing scenarios. We first evaluated which short-read technology was best suited for analyzing gene-rich genomic regions in Arabidopsis and determined the minimum sequencing depth required to confidently call single nucleotide variants. We also designed ways to discriminate mutagenesis-induced mutations from background Single Nucleotide Polymorphisms in mutants isolated in Arabidopsis non-reference lines. In addition, we simulated bulked segregant mapping populations for identifying point mutations and monitored how the size of the mapping population and the sequencing depth affect mapping precision. Finally, we provide the computational basis of a protocol that we already used to map T-DNA insertions with paired-end Illumina-like reads, using very low sequencing depths and pooling several mutants together; this approach can also be used with single-end reads as well as to map any other insertional mutagen. All these simulations proved useful for designing experiments that allowed us to map several mutations in Arabidopsis.

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Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis

Bioscience Reports ◽

10.1042/bsr20192933 ◽

2020 ◽

Vol 40 (5) ◽

Author(s):

Jindong Hao ◽

Chengshun Li ◽

Chao Lin ◽

Yang Hao ◽

Xianfeng Yu ◽

...

Keyword(s):

Gene Expression ◽

Cell Apoptosis ◽

Epigenetic Modification ◽

Point Mutations ◽

Base Editing ◽

Non Coding Rna ◽

Induce Cell Apoptosis ◽

The Relationship ◽

Adenine Base

Abstract Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T–A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.

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