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

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

Nucleic Acids Research ◽

10.1093/nar/gkaa215 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W340-W347 ◽

Cited By ~ 1

Author(s):

Roy Rabinowitz ◽

Shiran Abadi ◽

Shiri Almog ◽

Daniel Offen

Keyword(s):

Binding Site ◽

Genome Editing ◽

Point Mutation ◽

Point Mutations ◽

Computational Tool ◽

Coding Sequences ◽

Coding Sequence ◽

Base Editing ◽

A Genome ◽

Wide Range

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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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 but not adenine base editor generates mutations in mice

10.1101/731927 ◽

2019 ◽

Cited By ~ 1

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.

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Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d

Biotechnology and Bioengineering ◽

10.1002/bit.27813 ◽

2021 ◽

Author(s):

Xin Qiao ◽

Yanmin Gao ◽

Jiaojiao Li ◽

Zhaoguan Wang ◽

Hongyan Qiao ◽

...

Keyword(s):

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Single Nucleotide ◽

Sensitive Analysis

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Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽

10.1128/mbio.00342-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhiwei Hu ◽

Yannan Wang ◽

Qian Liu ◽

Yan Qiu ◽

Zhiyu Zhong ◽

...

Keyword(s):

Genome Editing ◽

Dna Breaks ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rna ◽

Guide Rnas ◽

Double Stranded Dna ◽

Target Sites ◽

Guide Sequence ◽

Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

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Annotate-it: a Swiss-knife approach to annotation, analysis and interpretation of single nucleotide variation in human disease

Genome Medicine ◽

10.1186/gm374 ◽

2012 ◽

Vol 4 (9) ◽

pp. 73 ◽

Cited By ~ 25

Author(s):

Alejandro Sifrim ◽

Jeroen KJ Van Houdt ◽

Leon-Charles Tranchevent ◽

Beata Nowakowska ◽

Ryo Sakai ◽

...

Keyword(s):

Human Disease ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Single Nucleotide

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Single Nucleotide Variation Analysis in 65 Candidate Genes for CNS Disorders in a Representative Sample of the European Population

Genome Research ◽

10.1101/gr.1299703 ◽

2003 ◽

Vol 13 (10) ◽

pp. 2271-2276 ◽

Cited By ~ 58

Author(s):

Y. Freudenberg-Hua

Keyword(s):

Candidate Genes ◽

Representative Sample ◽

European Population ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Variation Analysis ◽

Single Nucleotide ◽

Cns Disorders

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Newborn screening and single nucleotide variation profiling of TSHR, TPO, TG and DUOX2 candidate genes for congenital hypothyroidism

Molecular Biology Reports ◽

10.1007/s11033-020-05803-x ◽

2020 ◽

Vol 47 (10) ◽

pp. 7467-7475

Author(s):

Yedukondalu Kollati ◽

Radha Rama Devi Akella ◽

Shaik Mohammad Naushad ◽

Divya Borkar ◽

Maunika Thalla ◽

...

Keyword(s):

Newborn Screening ◽

Candidate Genes ◽

Congenital Hypothyroidism ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Single Nucleotide

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Association of Functional Single Nucleotide Variation in the NLRP3 Gene with Survival Outcomes After Unrelated Bone Marrow Transplantation.

Blood ◽

10.1182/blood.v120.21.3140.3140 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3140-3140

Author(s):

Akiyoshi Takami ◽

J. Luis Espinoza ◽

Keitaro Matsuo ◽

Yasuo Morishima ◽

Makoto Onizuka ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Single Nucleotide ◽

Hematopoietic Stem ◽

Nlrp3 Gene ◽

Transplant Outcomes ◽

Functional Relevance ◽

The Impact

Abstract Abstract 3140 NLRP3 is an intracellular trigger of IL-1β production that plays important roles in the regulation of inflammation and apoptosis. A single nucleotide variation in the 3'-untranslated region of the NLRP3 gene, rs10754558 (+29940G>C), is linked to several immunological diseases. When we examined the impact of the NLRP3 genotype in a cohort consisting of 392 pairs of patients with hematologic malignancies and their unrelated HLA 12/12 matched bone marrow donors transplanted through the Japan Donor Marrow Program, the recipient NLRP3 GG genotype was found to be associated with a significantly worse 5-year overall survival (OS) rate (34% vs. 50%, P=0.006) (Fig. 1) and a trend toward a higher transplant-related mortality (TRM) rate (39% vs. 27%, P=0.09) than the recipient CC or CG genotype. The recipient GG genotype remained statistically significant in the multivariate analysis for OS (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.22 to 2.22; P=0.004) and TRM (HR, 2.28; 95% CI, 1.20 to 4.35; P=0.01). The donor NLRP3 genotype did not significantly influence the transplant outcomes. Next, we investigated the functional relevance of the NLRP3 +29940G>C variant. When leukocytes from healthy individuals were stimulated in vitro with NLRP3 ligand, the leukocytes with the NLRP3 GG genotype produced significantly more IL-1β than those with the NLRP3 CC or CG genotype (Fig. 2). These findings substantiate the functional relevance of the NLRP3 variant, and suggest that the higher IL-1β secretion in the peri-transplant period by recipients with the NLRP3 GG genotype likely accounts for their poor transplant outcomes. NLRP3 genotyping could therefore be useful in predicting prognoses and creating therapeutic strategies for improving the final outcomes of patients who undergo allogeneic hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

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