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Analysis and minimization of cellular RNA editing by DNA adenine base editors

Published on May 1, 2019in Science Advances11.5
· DOI :10.1126/sciadv.aax5717
Holly A. Rees10
Estimated H-index: 10
(Broad Institute),
Christopher Wilson3
Estimated H-index: 3
(Broad Institute)
+ 1 AuthorsDavid R. Liu73
Estimated H-index: 73
(Broad Institute)
Sources
Abstract
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 both deaminase domains, we developed new ABE variants that retain their ability to edit DNA efficiently but show greatly reduced RNA editing activity, as well as lower off-target DNA editing activity and reduced indel by-product formation, in three mammalian cell lines. By decoupling DNA and RNA editing activities, these ABE variants increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity.
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References31
Newest
#1Chun-Qing Song (UMMS: University of Massachusetts Medical School)H-Index: 13
#2Tingting Jiang (UMMS: University of Massachusetts Medical School)H-Index: 2
Last. Wen XueH-Index: 28
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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 ...
3 CitationsSource
#1Daesik Kim (SNU: Seoul National University)H-Index: 14
#2Da-eun Kim (SNU: Seoul National University)H-Index: 3
Last. Jin-Soo Kim (SNU: Seoul National University)H-Index: 53
view all 5 authors...
Adenine base editors1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants1–4. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing5–8, to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites co...
14 CitationsSource
#1Kendell Clement (Broad Institute)H-Index: 16
#2Holly A. Rees (Broad Institute)H-Index: 10
Last. Luca Pinello (Broad Institute)H-Index: 22
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18 CitationsSource
#1Shuai Jin (CAS: Chinese Academy of Sciences)H-Index: 3
#2Yuan Zong (CAS: Chinese Academy of Sciences)H-Index: 7
Last. Caixia Gao (CAS: Chinese Academy of Sciences)H-Index: 27
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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→...
55 CitationsSource
#1Erwei Zuo (CAS: Chinese Academy of Sciences)H-Index: 6
#2Yidi Sun (CAS-MPG Partner Institute for Computational Biology)H-Index: 8
Last. Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
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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 bl...
66 CitationsSource
#1Puping Liang (SYSU: Sun Yat-sen University)H-Index: 8
#2Xiaowei Xie (SYSU: Sun Yat-sen University)H-Index: 7
Last. Zhou SongyangH-Index: 45
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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) o...
10 CitationsSource
#1Jeffrey J. Ishizuka (MIT: Massachusetts Institute of Technology)H-Index: 2
#2Robert T. Manguso (MIT: Massachusetts Institute of Technology)H-Index: 5
Last. W. Nicholas Haining (MIT: Massachusetts Institute of Technology)H-Index: 34
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Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and...
33 CitationsSource
#1Wei Qin (PKU: Peking University)H-Index: 5
#2Xiaochan Lu (PKU: Peking University)H-Index: 3
Last. Shuo Lin (UCLA: University of California, Los Angeles)H-Index: 53
view all 6 authors...
Base editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies. Recently, adenine base editors (ABEs) have been developed to mediate the conversion of A•T to G•C in genomic DNA of human cells, mice, and plants. Here, we investigated the activity and efficiency of several adenine base editors in zebrafish and showed that base editing can...
2 CitationsSource
#1Hye-Kyung Lee (NIH: National Institutes of Health)H-Index: 13
#2Michaela Willi (NIH: National Institutes of Health)H-Index: 8
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
view all 7 authors...
Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyze on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more...
12 CitationsSource
#1Holly A. Rees (Broad Institute)H-Index: 10
#2David R. Liu (Broad Institute)H-Index: 73
The originally published article contained errors in reference numbering throughout table 1 (DNA base editors and their approximate editing windows) due to the unintended propagation of reference numbering from an earlier version of the table. The article has now been corrected online. The editors apologize for this error.
3 CitationsSource
Cited By10
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Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; res...
1 CitationsSource
#1Xiao Wang (CAS: Chinese Academy of Sciences)H-Index: 9
#2Chengfeng Ding (CAS: Chinese Academy of Sciences)
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Summary The advent of base editors (BEs) holds great potential for correcting pathogenic-related point mutations to treat relevant diseases. However, Cas9 nickase (nCas9)-derived BEs lead to DNA double-strand breaks, which can trigger unwanted DNA damage response (DDR). Here, we show that the original version of catalytically dead Cas12a (dCas12a)-conjugated BEs induce a basal level of DNA breaks and minimally activate DDR proteins, including H2AX, ATM, ATR, and p53. By fusing dCas12a with engin...
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#2Soh Ishiguro (UTokyo: University of Tokyo)H-Index: 2
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We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors. Base editors that modify both adenosine and cytosine broaden ...
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Muscular dystrophies are debilitating disorders that result in progressive weakness and degeneration of skeletal muscle. Although the genetic mutations and clinical abnormalities of a variety of neuromuscular diseases are well known, no curative therapies have been developed to date. The advent of genome editing technology provides new opportunities to correct the underlying mutations responsible for many monogenic neuromuscular diseases. For example, Duchenne muscular dystrophy, which is caused...
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Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator-Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost-effectiveness. Various classes of CRISPR/Cas systems have been developed, bu...
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Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, ...
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