Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis

Zhou, Chunmei; Sun, Yidi; Yan, Rui; Liu, Yajing; Zuo, Erwei; Gu, Chan; Han, Linxiao; Wu, Yu; Hu, Xin; Zeng, Rong; Li, Yixue; Zhou, Haibo; Guo, Fan; Yang, Hui

doi:10.1038/s41586-019-1314-0

Cited by 341 publications

(303 citation statements)

References 40 publications

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“…To generate data on all the constructs of interest in a high-throughput manner, we began with targeted amplification and high throughput sequencing of two cellular RNAs in HEK293T cells treated with ABEs. Consistent with previous publications 16,17,40,41 treatment with ABE7, and, to a greater extent, ABE8 lead to detectable A-to-I deamination in cellular RNA when delivered by plasmid overexpression ( Supplementary Fig 24e). However, when delivered as an mRNA construct, the level of mRNA deamination was reduced by an average of 34-fold (in the case of ABE7.10-d) to 134-fold (ABE8.17-m) ( Supplementary Fig.…”

Section: Off Target Analyses Of Abe8s In Mammalian Cellssupporting

confidence: 91%

“…As with all base editors, ABE8s have the potential to act at off-target loci in the genome and transcriptome 1,2,16,17,[36][37][38][39][40][41] . Guided by prior publications on this topic, we undertook an extensive assessment of the off-target deamination effects of ABE8s.…”

Section: Off Target Analyses Of Abe8s In Mammalian Cellsmentioning

confidence: 99%

“…We began by assessing whether previously-published mutations 16,17,40,41 , designed to mitigate spurious cellular RNA deamination for ABE7.10 is also compatible with our ABE8s (we chose ABE8.17-m to probe this question). All of the installed RNA off-target minimizing mutations decreased the on-target editing frequencies of ABE8.17-m to differing extents, with V106W 17 and F148A 41 impairing ABE8 the least ( Supplementary Fig.…”

Section: Off Target Analyses Of Abe8s In Mammalian Cellsmentioning

confidence: 99%

“…Next, we measured the levels of spurious deamination of cellular mRNA 16,17,40,41 associated with ABE8s. To generate data on all the constructs of interest in a high-throughput manner, we began with targeted amplification and high throughput sequencing of two cellular RNAs in HEK293T cells treated with ABEs.…”

Section: Off Target Analyses Of Abe8s In Mammalian Cellsmentioning

confidence: 99%

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Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli

Lam

Rees

et al. 2020

Preprint

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The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ∼1.5x higher editing at protospacer positions A5-A7 and ∼3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ∼4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

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Section: Off Target Analyses Of Abe8s In Mammalian Cellssupporting

confidence: 91%

Section: Off Target Analyses Of Abe8s In Mammalian Cellsmentioning

confidence: 99%

Section: Off Target Analyses Of Abe8s In Mammalian Cellsmentioning

confidence: 99%

Section: Off Target Analyses Of Abe8s In Mammalian Cellsmentioning

confidence: 99%

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Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli

Lam

Rees

et al. 2020

Preprint

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“…Zhou et al quantitatively evaluated single nucleotide variations (SNVs) that have been induced by CBEs or ABEs. The result show that these two systems can generate considerable off‐target SNVs in RNA sequences.…”

Section: Strategies To Increase Target Editing Efficiency and Avoid Omentioning

confidence: 99%

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

et al. 2020

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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, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized.

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Specificity of CRISPR‐Cas9 Gene Editing

Selfjord¹,

Madsen²,

Akçakaya³

2022

Genome Editing in Drug Discovery

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Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis

Cited by 341 publications

References 40 publications

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

Specificity of CRISPR‐Cas9 Gene Editing

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