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High-fidelity base editor with no detectable genome-wide off-target effects

Published on Feb 9, 2020in bioRxiv
· DOI :10.1101/2020.02.07.939074
Erwei Zuo7
Estimated H-index: 7
(CAS: Chinese Academy of Sciences),
Yidi Sun8
Estimated H-index: 8
(CAS: Chinese Academy of Sciences)
+ 8 AuthorsLIYixue59
Estimated H-index: 59
Abstract
Base editors hold promise for correcting pathogenic mutations, while substantial single nucleotide variations (SNVs) on both DNA and RNA were generated by cytosine base editors (CBEs). Here we examined possibilities to reduce off-target effects by engineering cytosine deaminases. By screening 24 CBEs harboring various rAPOBEC1 (BE3) or human APOBEC3A (BE3-hA3A) mutations on the ssDNA or RNA binding domain, we found 8 CBE variations could maintain high on-target editing efficiency. Using Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) method and RNA sequencing analysis, we found DNA off-target SNVs induced by BE3 could be completely eliminated in BE3R126E but the off-target RNA SNVs was only slightly reduced. By contrast, BE3-hA3AY130F abolished the RNA off-target effects while could not reduce the DNA off-target effects. Notably, BE3R132E, BE3W90Y+R126E and BE3W90F+R126E achieved the elimination of off-target SNVs on both DNA and RNA, suggesting the feasibility of engineering base editors for high fidelity deaminases.
  • References (30)
  • Citations (2)
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References30
Newest
#1Changyang Zhou (CAS: Chinese Academy of Sciences)H-Index: 5
#2Yidi Sun (CAS: Chinese Academy of Sciences)H-Index: 8
Last. Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
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Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1–3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4–8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9–13. For example, the cytosine deaminase APOBEC1—which is used...
16 CitationsSource
#1Julian Grünewald (Harvard University)H-Index: 2
#1Julian Grünewald (Harvard University)H-Index: 5
Last. J. Keith Joung (Harvard University)H-Index: 68
view all 7 authors...
Abstract CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-i...
7 CitationsSource
#1Julian GrünewaldH-Index: 2
#2Ronghao Zhou (Harvard University)H-Index: 3
Last. J. Keith JoungH-Index: 68
view all 7 authors...
CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications1,2. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex3,4. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells5,6. Here we show that a CBE with rAPOBEC1 can cause ...
27 CitationsSource
#1Shuai Jin (CAS: Chinese Academy of Sciences)H-Index: 4
#2Yuan Zong (CAS: Chinese Academy of Sciences)H-Index: 10
Last. Caixia Gao (CAS: Chinese Academy of Sciences)H-Index: 34
view all 11 authors...
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: 7
#2Yidi Sun (CAS-MPG Partner Institute for Computational Biology)H-Index: 8
Last. Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
view all 10 authors...
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
#1Hui YangH-Index: 46
#1Hui YangH-Index: 19
Last. Lars M. Steinmetz (Stanford University)H-Index: 60
view all 8 authors...
Genome editing tools including CRISPR/Cas9 and base editors hold great promise for correcting pathogenic mutations. Unbiased genome-wide off-target effects of the editing in mammalian cells is required before clinical applications, but determination of the extent of off-target effects has been difficult due to the existence of single nucleotide polymorphisms (SNPs) in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-...
8 CitationsSource
#1Holly A. Rees (Broad Institute)H-Index: 12
#2David R. Liu (Broad Institute)H-Index: 81
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
#1Holly A. Rees (Broad Institute)H-Index: 12
#2David R. Liu (Broad Institute)H-Index: 81
RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nuc...
75 CitationsSource
#1Xiao WangH-Index: 2
#2Jianan LiH-Index: 8
Last. Li YangH-Index: 38
view all 10 authors...
Increased efficiency of base editing in methylated DNA using human APOBEC3A as a deaminase.
31 CitationsSource
#1Jason M. GehrkeH-Index: 3
Last. J. Keith JoungH-Index: 68
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The precision of base editing is enhanced with an engineered version of the APOBEC3A deaminase.
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#1Wenhui Zhang (SCAU: South China Agricultural University)H-Index: 3
#2Tomomi Aida (McGovern Institute for Brain Research)H-Index: 1
Last. Duanduan Li (SCAU: South China Agricultural University)H-Index: 1
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Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity ...
1 CitationsSource
Improvements to a method known as base editing could pave the way for safer gene therapies. Improvements to a method known as base editing could pave the way for safer gene therapies.
Source