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Base editing generates substantial off-target single nucleotide variants

Published on Nov 27, 2018in bioRxiv
路 DOI :10.1101/480145
Hui Yang19
Estimated H-index: 19
,
LIYixue57
Estimated H-index: 57
+ 5 AuthorsLars M. Steinmetz56
Estimated H-index: 56
(Stanford University)
Abstract
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-target mutations without interference of SNPs. We applied GOTI to both the CRISPR-Cas9 and base editing (BE3) systems by editing one blastomere of the two-cell mouse embryo and then compared whole genome sequences of progeny-cell populations at E14.5 stage. Sequence analysis of edited and non-edited cell progenies showed that undesired off-target single nucleotide variants (SNVs) are rare (average 10.5) in CRISPR-edited mouse embryos, with a frequency close to the spontaneous mutation rate. By contrast, BE3 editing induced over 20-fold higher SNVs (average 283), raising the concern of using base-editing approaches for biomedical application.
  • References (40)
  • Citations (4)
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References40
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#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
#1Holly A. Rees (Broad Institute)H-Index: 10
#2David R. Liu (Broad Institute)H-Index: 73
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
#1Michaela Willi (NIH: National Institutes of Health)H-Index: 8
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9 CitationsSource
#1Huiyun SeoH-Index: 1
#2Jin-Soo Kim (SNU: Seoul National University)H-Index: 53
Base editors function in mouse fetuses and in the livers of adult mice to treat a genetic disorder.
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#1Robert M. Yarrington (UofU: University of Utah)H-Index: 7
#2Surbhi Verma (UofU: University of Utah)H-Index: 6
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Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin st...
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Cas9-induced double stranded breaks can cause large deletions near the target site and more complex genomic rearrangements in mouse and human stem cells.
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BACKGROUND Nearly five decades ago, visionary scientists hypothesized that genetic modification by exogenous DNA might be an effective treatment for inherited human diseases. This 鈥済ene therapy鈥 strategy offered the theoretical advantage that a durable and possibly curative clinical benefit would be achieved by a single treatment. Although the journey from concept to clinical application has been long and tortuous, gene therapy is now bringing new treatment options to multiple fields of medicine...
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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-w...
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The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. To address this, we provide three resources. First, we introduce Cassiopeia - a suite of scalable and theoretically grounded maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for eval...
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Gene editing tools are being rapidly developed, accelerating many areas of cell and gene therapy research. Each successive gene editing technology promises increased efficacy, improved specificity, reduced manufacturing cost and design complexity; all of which are currently epitomised by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9) platform. Since its conceptualisation, CRISPR-based gene editing has been applied to existing methodolo...
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Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)鈥揳ssociated protein 9 (Cas9) nickase, enable the efficien...
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Editing the genome of human embryos is ethically fraught. But some projects show how diligent, ethical work can grow the gene-editing field.
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Genome editing is revolutionising our ability to modify genomes with exquisite precision for medical and agricultural applications, and in basic research. The first International Summit on Human Genome Editing, organised jointly by the US National Academies of Sciences and Medicine, the Chinese Academy of Sciences and the UK Royal Society, was held in Washington DC at the end of 2015. Its aim was to explore scientific, legal and ethical perspectives on the prospective use of human genome editing...
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