scholarly journals Programmable Base Editing of the Sheep Genome Revealed No Genome-Wide Off-Target Mutations

2019 ◽  
Vol 10 ◽  
Author(s):  
Shiwei Zhou ◽  
Bei Cai ◽  
Chong He ◽  
Ying Wang ◽  
Qiang Ding ◽  
...  
Keyword(s):  
Base Editing ◽  
Sheep Genome ◽  
Genome Wide

scholarly journals Protocol for examining and eliminating base editor-induced genome-wide and transcriptome-wide off-target mutations

2021 ◽  
Author(s):  
Lijie Wang ◽  
Wei Xue ◽  
Hongxia Zhang ◽  
Runze Gao ◽  
Houyuan Qiu ◽  
...  
Keyword(s):  
Clinical Applications ◽  
Cytidine Deaminases ◽  
Base Editing ◽  
Plasmid Construction ◽  
Background Levels ◽  
Genome Wide ◽  
Deoxycytidine Deaminase

Abstract Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.

scholarly journals Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L)

2020 ◽  
Vol 2 ◽  
Author(s):  
Limin Hu ◽  
Olalekan Amoo ◽  
Qianqian Liu ◽  
Shengli Cai ◽  
Miaoshan Zhu ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Molecular Breeding ◽  
Target Genes ◽  
Agronomic Traits ◽  
Crop Improvement ◽  
Brassica Napus L ◽  
Editing Efficiency ◽  
Base Editing ◽  
Expected Gain ◽  
Genome Wide

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

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 Prime editing for functional repair in patient-derived disease models

2020 ◽  
Author(s):  
Imre F. Schene ◽  
Indi P. Joore ◽  
Rurika Oka ◽  
Michal Mokry ◽  
Anke H.M. van Vugt ◽  
...  
Keyword(s):  
Adult Stem Cells ◽  
Gene Editing ◽  
Therapeutic Potential ◽  
Gene Encoding ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Genome Wide ◽  
Culture Models ◽  
Liver Organoids ◽  
Target Effects

AbstractPrime editing is a novel genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. We develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers diseasecausing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

scholarly journals Prime editing for functional repair in patient-derived disease models

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Imre F. Schene ◽  
Indi P. Joore ◽  
Rurika Oka ◽  
Michal Mokry ◽  
Anke H. M. van Vugt ◽  
...  
Keyword(s):  
Adult Stem Cells ◽  
Gene Editing ◽  
Therapeutic Potential ◽  
Gene Encoding ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Genome Wide ◽  
Culture Models ◽  
Liver Organoids ◽  
Target Effects

Abstract Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

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 Multiplex base editing to convert TAG into TAA codons in the human genome

2021 ◽  
Author(s):  
Yuting Chen ◽  
Eriona Hysolli ◽  
Anlu Chen ◽  
Stephen Casper ◽  
Songlei Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Genome ◽  
Mammalian Cells ◽  
Large Scale ◽  
Essential Gene ◽  
Human Cells ◽  
Viral Resistance ◽  
Computational Tool ◽  
Base Editing ◽  
Genome Wide

Large-scale recoding has been shown to enable novel amino acids, biocontainment and viral resistance in bacteria only so far. Here we extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~ 40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and multiplex editing in mammalian cells.

scholarly journals Perturbing proteomes at single residue resolution using base editing

2019 ◽  
Author(s):  
Philippe C Després ◽  
Alexandre K Dubé ◽  
Motoaki Seki ◽  
Nozomu Yachie ◽  
Christian R Landry
Keyword(s):  
Cytidine Deaminase ◽  
Amino Acid Residues ◽  
Single Experiment ◽  
Base Editing ◽  
Genome Wide ◽  
A Genome ◽  
Key Features ◽  
Dna Editing ◽  
Genome Scale ◽  
Protein Variants

AbstractBase editors derived from CRISPR-Cas9 systems and DNA editing enzymes offer an unprecedented opportunity for the precise modification of genes, but have yet to be used at a genome-scale throughput. Here, we test the ability of an editor based on a cytidine deaminase, the Target-AID base editor, to systematically modify genes genome-wide using the set of yeast essential genes. We tested the effect of mutating around 17,000 individual sites in parallel across more than 1,500 genes in a single experiment. We identified over 1,100 sites at which mutations have a significant impact on fitness. Using previously determined and preferred Target-AID mutational outcomes, we predicted the protein variants caused by each of these gRNAs. We found that gRNAs with significant effects on fitness are enriched in variants predicted to be deleterious by independent methods based on site conservation and predicted protein destabilization. Finally, we identify key features to design effective gRNAs in the context of base editing. Our results show that base editing is a powerful tool to identify key amino acid residues at the scale of proteomes.

scholarly journals beditor: A computational workflow for designing libraries of guide RNAs for CRISPR-mediated base editing

2018 ◽  
Author(s):  
Rohan Dandage ◽  
Philippe C Després ◽  
Nozomu Yachie ◽  
Christian R Landry
Keyword(s):  
A Priori ◽  
Maximum Activity ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genome Wide ◽  
Disease Mutations ◽  
Computational Workflow ◽  
Activity Window ◽  
Target Effects

ABSTRACTCRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, PAM recognition sequences and genomes of many species. Additionally, in order to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates, called beditor scores are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces non-confounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base-editing to create or remove thousands of clinically important human disease mutations.

scholarly journals Erratum: Precise genome-wide base editing by the CRISPR Nickase system in yeast

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Atsushi Satomura ◽  
Ryosuke Nishioka ◽  
Hitoshi Mori ◽  
Kosuke Sato ◽  
Kouichi Kuroda ◽  
...  
Keyword(s):  
Base Editing ◽  
Genome Wide
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