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Genome-wide profiling of adenine base editor specificity by EndoV-seq

Published on Jan 8, 2019in Nature Communications11.88
· DOI :10.1038/s41467-018-07988-z
Puping Liang7
Estimated H-index: 7
(SYSU: Sun Yat-sen University),
Xiaowei Xie6
Estimated H-index: 6
(SYSU: Sun Yat-sen University)
+ 9 AuthorsZhou Songyang43
Estimated H-index: 43
Cite
Abstract
The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2–19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7–320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease.
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  • References (59)
  • Citations (8)
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References59
Newest
Published on Dec 1, 2018in Cell discovery
Yuanwu Ma6
Estimated H-index: 6
(Academy of Medical Sciences, United Kingdom),
Lei Yu70
Estimated H-index: 70
(Academy of Medical Sciences, United Kingdom)
+ 10 AuthorsShuo Pan3
Estimated H-index: 3
(Academy of Medical Sciences, United Kingdom)
Published on Dec 1, 2018in Nature Communications11.88
Zhen Liu11
Estimated H-index: 11
(CAS: Chinese Academy of Sciences),
Zongyang Lu3
Estimated H-index: 3
(ShanghaiTech University)
+ 10 AuthorsYu Zhang4
Estimated H-index: 4
(ShanghaiTech University)
A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to...
Published on Sep 1, 2018in Protein & Cell7.58
Puping Liang7
Estimated H-index: 7
(SYSU: Sun Yat-sen University),
Hongwei Sun4
Estimated H-index: 4
(SYSU: Sun Yat-sen University)
+ 10 AuthorsWenbin Ma12
Estimated H-index: 12
(SYSU: Sun Yat-sen University)
Published on Sep 1, 2018in Protein & Cell7.58
Lei Yang2
Estimated H-index: 2
(ECNU: East China Normal University),
Xiaohui Zhang2
Estimated H-index: 2
(ECNU: East China Normal University)
+ 16 AuthorsYu Wei1
Estimated H-index: 1
(ECNU: East China Normal University)
Published on Apr 27, 2018in Nature Biotechnology31.86
Seuk-Min Ryu5
Estimated H-index: 5
,
Taeyoung Koo14
Estimated H-index: 14
+ 8 AuthorsJin-Soo Kim52
Estimated H-index: 52
Adenine base editing is used to treat a mouse model of Duchenne muscular dystrophy and to create defined mutations in mouse embryos.
Published on Apr 1, 2018in Nature43.07
Johnny H. Hu7
Estimated H-index: 7
,
Shannon M. Miller2
Estimated H-index: 2
+ 8 AuthorsZhi Lin1
Estimated H-index: 1
Phage-assisted continuous evolution of Cas9 variants with broad PAM compatibility and high DNA specificity that can be used for transcriptional activation, gene disruption and base editing.
Published on Jan 29, 2018in Nature Biotechnology31.86
Antonio Casini5
Estimated H-index: 5
,
Michele Olivieri2
Estimated H-index: 2
+ 9 AuthorsFrancesca Demichelis53
Estimated H-index: 53
Evolved SpCas9 variant evoCas9 has improved specificity and retains near wild-type on-target activity.
Published on Mar 1, 2018in Nature plants13.30
Jin-Soo Kim52
Estimated H-index: 52
(SNU: Seoul National University)
Adenine base editors (ABEs), composed of an engineered deaminase and a catalytically impaired CRISPR–Cas9 variant, are powerful new tools for targeted base editing in cells and organisms. Together with cytosine base editors (CBEs), ABEs enable single-nucleotide conversions cleanly, efficiently and reversibly without double-stranded DNA cleavage, advancing genome editing in a new dimension.
Published on Dec 1, 2017in Nature Communications11.88
Yihan Zhang4
Estimated H-index: 4
(PKU: Peking University),
Wei Qin7
Estimated H-index: 7
(PKU: Peking University)
+ 5 AuthorsShuo Lin52
Estimated H-index: 52
(PKU: Peking University)
Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable “base editing” system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5′-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be ...
Published on Nov 1, 2017in Protein & Cell7.58
Puping Liang7
Estimated H-index: 7
(SYSU: Sun Yat-sen University),
Chenhui Ding14
Estimated H-index: 14
(SYSU: Sun Yat-sen University)
+ 13 AuthorsYongxiang Liu1
Estimated H-index: 1
(SYSU: Sun Yat-sen University)
β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogen...
Cited By8
Newest
Published on Sep 23, 2019in Nature Biotechnology31.86
Heon Seok Kim6
Estimated H-index: 6
,
You Kyeong Jeong1
Estimated H-index: 1
(Hanyang University)
+ 2 AuthorsSangsu Bae13
Estimated H-index: 13
(Hanyang University)
Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5–7) and in a confined TC*N sequence context. Adenine base editor–induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-speci...
Published on 2019in Molecular Therapy8.40
Choongil Lee5
Estimated H-index: 5
(SNU: Seoul National University),
Dong Hyun Jo12
Estimated H-index: 12
(Seoul National University Hospital)
+ 5 AuthorsSangsu Bae13
Estimated H-index: 13
(Hanyang University)
A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to ...
Published on Jul 23, 2019in bioRxiv
Ruisha Hong (SYSU: Sun Yat-sen University), Sidi Ma (SYSU: Sun Yat-sen University), Feng Wang (SYSU: Sun Yat-sen University)
Adenine base editor (ABE) mediates the conversion of A to G in genomic DNA. In human, approximately 47.8% of known pathogenic SNPs can be corrected by A to G conversion, indicating that ABE have tremendous potential in gene therapy. However, the off-target activity of ABE limits its clinical application. ABE off-target activity in DNA is depended on the bonding of Streptococcus pyogenes Cas9 (SpCas9) on off-target sites [1, 2]. Therefore, using high-fidelity Cas9 should be able to improve the sp...
Published on Apr 30, 2019in Cell Biology and Toxicology5.10
Puping Liang7
Estimated H-index: 7
(SYSU: Sun Yat-sen University),
Puping Liang + -1 AuthorsJunjiu Huang
Published on May 21, 2019in Genes3.33
Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CFTR gene. There are at least 346 disease-causing variants in the CFTR gene, but effective small-molecule therapies exist for only ~10% of them. One option to treat all mutations is CFTR cDNA-based therapy, but clinical trials to date have only been able to stabilise rather than improve lung function disease in patients. While cDNA-based therapy is already a clinical reality for a number of diseases, some...
Published on May 1, 2019in Science Advances
Holly A. Rees8
Estimated H-index: 8
(Broad Institute),
Christopher Wilson1
Estimated H-index: 1
(Broad Institute)
+ 1 AuthorsDavid R. Liu70
Estimated H-index: 70
(Broad Institute)
Adenine base editors (ABEs) enable precise and efficient conversion of target A∙T base pairs to G·C base pairs in genomic DNA with a minimum of by-products. While ABEs have been reported to exhibit minimal off-target DNA editing, off-target editing of cellular RNA by ABEs has not been examined in depth. Here, we demonstrate that a current ABE generates low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in b...
Published on May 1, 2019in Cell Research17.85
Guigen Zhang (PKU: Peking University), Guigen Zhang1
Estimated H-index: 1
(PKU: Peking University)
+ 0 AuthorsWensheng Wei16
Estimated H-index: 16
(PKU: Peking University)
Published on Feb 28, 2019in Science41.04
Shuai Jin3
Estimated H-index: 3
(CAS: Chinese Academy of Sciences),
Yuan Zong6
Estimated H-index: 6
(CAS: Chinese Academy of Sciences)
+ 8 AuthorsFeng Zhang104
Estimated H-index: 104
(UMN: University of Minnesota)
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→...
Published on Feb 25, 2019in Nature Biomedical Engineering
Chun-Qing Song11
Estimated H-index: 11
(UMMS: University of Massachusetts Medical School),
Tingting Jiang2
Estimated H-index: 2
(UMMS: University of Massachusetts Medical School)
+ 12 AuthorsLukas E. Dow23
Estimated H-index: 23
(Cornell University)
In contrast to traditional CRISPR–Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight ...
View next paperProgrammable editing of a target base in genomic DNA without double-stranded DNA cleavage