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Targeted Manipulation of Cellular RNA m6A Methylation at Single Base Level Using an Engineered dCas13b-METTL14 Fusion Protein

ABSTRACT: We engineered m6A methyltransferase with dCas13-M14 to achieve site-specific editing of m6A. The resultant m6A editors can be programmed with a guide RNA, allowing functional comparison of single site methylation in different mRNA regions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE185755 | GEO | 2024/10/12

REPOSITORIES: GEO

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Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates

Project description:We engineered m6A methyltransferase or demethylases with CRISPR-Cas9 to achieve site-specific editing of m6A. The resultant m6A editors can be programmed with a guide RNA, allowing functional comparison of single site methylation in different mRNA regions.

2019-06-01 | GSE132051 | GEO

Transcriptome wide edits after split-engineered base editor treatment

Project description:We report transcriptome wide edits comparison between split-engineered base editors and intact base editors. Our results show that, split-engineered base editors show backgound levels of unique C>U edits when compared to intact base editors.

2021-10-18 | GSE181109 | GEO

Genome-wide identification of N6-methyladenosine by microarray

Project description:Although internal PolyA RNA modification N6-methyladenosine (m6A) plays essential roles in diverse biological processes, technology to detect precise m6A sites at transcriptome-wide scale is lacking. Here, we discovered that m6A interferes A (Adenine) – U (Uracil) or A-T (Thymidine) pairing. Based on differential hybridization between methylated vs. unmethylated RNAs to a DNA probe, we developed tiling microarray to pinpoint m6A sites in mouse transcriptome. We validated some of the identified sites and provided evidence to suggest that one functional mechanism of m6A is to block small RNA targeting to methylated mRNA. We designed a custom tiling array with to examine the precise location of m6A within meRIP-seq peaks from mouse embryonic stem cells determined in our previous publication (Wang et al., 2014). Each custom two-channel Agilent tiling array harbors 947,952 probes. Each probe is 25 nucleotides (nt), and any two adjacent probes in the genomic coordinate overlap each other by 19 nt. The Cy5 or red channel corresponds to Mettl14 knockout (M14) or DZA mutant mESC cell line, and Cy3 or green channel is associated with wild type cell line treated with scramble hairpin (SCR). Thus, in principle a higher Cy5/Cy3 signal for each probe reflects an increased hybridization to the oligonucleotide due to de-methylation of a particular RNA molecule in M14 or DZA condition relative to the SCR control. Moreover, we employed additional arrays with both channels dedicated for M14 as an external control for technical difference between the Cy5 and Cy3 dye (details below). For each comparison, we have three biological replicates, and therefore there are 9 tiling arrays in total (i.e., 3 arrays for M14 vs SCR, 3 arrays for DZA vs SCR, and 3 arrays for M14 vs M14).

2015-09-08 | E-GEOD-62530 | biostudies-arrayexpress

Targeted Manipulation of Cellular RNA m6A Methylation at Single Base Level Using an Engineered dCas13b-METTL14 Fusion Protein

Project description:Targeted Manipulation of Cellular RNA m6A Methylation at Single Base Level Using an Engineered dCas13b-METTL14 Fusion Protein

| PRJNA770674 | ENA

Base editing with a Cpf1–cytidine deaminase fusion

Project description:The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich PAM sequences. To overcome this limitation, we developed a CRISPR/Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and converts C to T in human cells with low levels of indels, non-C-to-T substitutions and off-target editing.

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High-fidelity base editing mediated by Cpf1-cytidine deaminase fusion

Project description:C-to-T base editing mediated by CRISPR/Cas9 base editors (BEs) needs a G/C-rich PAM and the editing fidelity is compromised by unwanted indels and non-C-to-T substitutions. We developed CRISPR/Cpf1-based BEs to recognize a T-rich PAM and induce efficient C-to-T editing with few indels and/or non-C-to-T substitutions. The requirement of editing fidelity in therapeutic-related trials necessitates the development of CRISPR/Cpf1-based BEs, which also facilitates base editing in A/T-rich regions.

2018-10-16 | GSE105002 | GEO

Cancer Mutations Rewire the RNA Methylation Specificity of METTL3-METTL14

Project description:Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N6-methyladenosine (m6A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to numerous cancers. Here we show that m6A modification location, rather than the overall modification level, can impact oncogenesis. A gain-of-function missense mutation found in cancer patients, METTL14R298P, promotes malignant cell growth in culture and in transgenic mice. The mutant methyltransferase preferentially modifies noncanonical sites and transforms gene expression without increasing global m6A levels in mRNAs. The altered substrate specificity is intrinsic to METTL3-METTL14, helping us to propose a structural model for how the METTL3-METTL14 complex detects RNA sequences. Together, our work highlights that m6A location is important for function and that noncanonical methylation sites may impact aberrant gene expression and oncogenesis.

2024-12-21 | PXD039447 | Pride

Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens

Project description:N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.

2021-10-21 | GSE179980 | GEO

Single base mapping of m6A by an antibody-independent method

Project description:N6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, related to pivotal RNA metabolism processes. The most popular high-throughput m6A identification method relies on the commercial m6A antibody but suffers from poor reproducibility and limited resolution. Exact location of m6A site is of great vital for understanding the dynamics, functions and machinery of RNA methylation. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic single base m6A map generated by m6A-REF-seq displayed a typical distribution pattern with enrichment adjacent to stop codon. Ligase-based and qPCR validation methods were used to confirm the individual m6A sites and quantify the methylation level, reinforcing the high accuracy of m6A-REF-seq. We applied m6A-REF-seq on five tissues from three mammals, showing that m6A sites were conserved and tend to gather together among species. (m6A-REF-seq had been named as Aim-seq.)

2019-07-03 | GSE125240 | GEO

Structure and Thermodynamics of N6-methyladenosine in RNA: a Spring-Loaded Base Modification

Project description:We determine m6A modification sites in GM12878 cells. Examination of m6A modification sites on the transcriptome of GM12878 cells, using a m6A-specific antibody.

2015-01-29 | E-GEOD-65380 | biostudies-arrayexpress

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