Dataset Information

Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis.

ABSTRACT: Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T-A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.

SUBMITTER: Hao J

PROVIDER: S-EPMC7201566 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis.

Hao Jindong J Li Chengshun C Lin Chao C Hao Yang Y Yu Xianfeng X Xia Yidan Y Gao Fei F Jiang Ziping Z Wang Dongxu D

Bioscience reports 20200501 5

Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cell ...[more]

PMID: 32323721

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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

Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis.

Publications

Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis.

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