Dataset Information

Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.

ABSTRACT:

Background

The base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration.

Results

In the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs containing A3A, A3A (Y130F), and rAPOBEC1(R33A) to compare their base editing efficiency in allotetraploid N. benthamiana (n = 4x). We used 14 target sites to compare their editing efficiency using transient transformation in tobacco plants. The sanger sequencing and deep sequencing results showed that A3A-CBE was the most efficient base editor. In addition, the results showed that A3A-CBE provided most comprehensive editing window (C₁ ~ C₁₇ could be edited) and had a better editing efficiency under the base background of TC. The target sites (T2 and T6) analysis in transformed N. benthamiana showed that only A3A-CBE can have C-to-T editing events and the editing efficiency of T2 was higher than T6. Additionally, no off-target events were found in transformed N. benthamiana.

Conclusions

All in all, we conclude that A3A-CBE is the most suitable vector for specific C to T conversion in N. benthamiana. Current findings will provide valuable insights into selecting an appropriate base editor for breeding polyploid plants.

SUBMITTER: Luo J

PROVIDER: S-EPMC10245509 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.

Luo Juan J Abid Muhammad M Tu Jing J Cai Xinxia X Zhang Yi Y Gao Puxin P Huang Hongwen H

BMC plant biology 20230607 1

<h4>Background</h4>The base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration.<h4>Results</h4>In the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs contain ...[more]

PMID: 37286962

Similar Datasets

Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities.

Dataset Information

Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.

Background

Results

Conclusions

Publications

Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.

Similar Datasets

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