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PLANT PHYSIOLOGY
Article . 2021 . Peer-reviewed
License: OUP Standard Publication Reuse
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Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor

Authors: Linnell Bentley Randall; Simon Sretenovic; Yuechao Wu; Desuo Yin; Tao Zhang; Joyce Van Eck; Yiping Qi;

Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor

Abstract

Abstract Cytosine base editors (CBEs) are the promising tools for precise genome editing in plants. It is important to investigate potential off-target effects of an efficient CBE at the genome and transcriptome levels in a major crop. Based on comparison of five cytidine deaminases and two different promoters for expressing single-guide RNAs (sgRNAs), we tested a highly efficient A3A/Y130F-BE3 system for efficient C-to-T base editing in tomato (Solanum lycopersicum). We then conducted whole-genome sequencing of four base-edited tomato plants, three Green fluorescent protein (GFP)-expressing control plants, and two wild-type plants. The sequencing depths ranged from 25× to 49× with read mapping rates >97%. No sgRNA-dependent off-target mutations were detected. Our data show an average of approximately 1,000 single-nucleotide variations (SNVs) and approximately 100 insertions and deletions (indels) per GFP control plant. Base-edited plants had on average elevated levels of SNVs (approximately 1,250) and indels (approximately 300) per plant. On average, about 200 more C-to-T (G-to-A) mutations were found in a base-edited plant than a GFP control plant, suggesting some level of sgRNA-independent off-target effects, though the difference is not statistically significant. We also conducted RNA sequencing of the same four base-edited plants and three GFP control plants. An average of approximately 200 RNA SNVs was discovered per plant for either base-edited or GFP control plants. Furthermore, no specific enrichment of C-to-U mutations can be found in the base-edited plants. Hence, we cannot find any evidence for bona fide off-target mutations by A3A/Y130F-BE3 at the transcriptome level.

Keywords

Cytosine, Solanum lycopersicum, Gene Expression Profiling, Genome-Wide Association Study, Plant Proteins

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
38
Top 10%
Top 10%
Top 10%
hybrid