Summary
N
6
-methyladenosine (m
6
A), the most prevalent internal RNA modification on mammalian messenger RNAs (mRNAs), regulates fates and functions of modified transcripts through m
6
A-specific binding proteins
1
–
5
. m
6
A is abundant in the nervous system and modulates various neural functions
6
–
11
. While m
6
A marks groups of mRNAs for coordinated degradation in various physiological processes
12
–
15
, the relevance of m
6
A in mRNA translation remains largely unknown
in vivo
. Here we show that, through its binding protein Ythdf1, m
6
A promotes protein synthesis of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of
Ythdf1
(
Ythdf1
-KO) exhibit learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Ythdf1 re-expression in the hippocampus of adult
Ythdf1
-KO mice rescues behavioral and synaptic defects, while hippocampus-specific acute knockdown of
Ythdf1
or
Mettl3
, the catalytic component of m
6
A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of Ythdf1 binding sites and m
6
A sites on hippocampal mRNAs uncovered key neuronal genes. Nascent protein labeling and tether reporter assays in hippocampal neurons revealed that Ythdf1 enhances protein synthesis in a neuronal-stimulus-dependent manner. Collectively, our results uncover a pathway of mRNA m
6
A methylation in learning and memory, which is mediated through Ythdf1 in response to stimuli.
The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich protospacer-adjacent motif (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 catalyzes C-to-T conversion in human cells, while inducing low levels of indels, non-C-to-T substitutions and off-target editing.
A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.
Highlights d BEACON induces basal levels of DNA breaks and DNA damage response d BEACON induces a basal level of RNA off-target mutations d BEACON induces in vivo base editing with high product purity
Summary
RNA splicing is related to many human diseases; however, lack of efficient genetic approaches to modulate splicing has prevented us from dissecting their functions in human diseases. Recently developed base editors (BEs) offer a new strategy to modulate RNA splicing by converting conservative splice sites, but it is limited by the editing precision and scope. To overcome the limitations of currently available BE-based tools, we combined SpCas9-NG with ABEmax to generate a new BE, ABEmax-NG. We demonstrated that ABEmax-NG performed precise A⋅T to G⋅C conversion with an expanded scope, thus covering many more splicing sites. Taking advantage of this tool, we precisely achieved A⋅T to G⋅C conversion exactly at the splice sites. We further modeled pathogenic RNA splicing
in vitro
and
in vivo
. Taken together, we successfully generated a versatile tool suitable for precise and broad editing at the splice sites.
Highlights d Base editing introduces substitutions at multiple histone H3 loci d Both H3R17H substitution and Carm1 truncation disrupt early embryo development d H3R17H substitution and Carm1 truncation lead to similar transcriptomic alterations d H3R17me regulates embryonic development through Yap1 and cell cycle signaling pathways
Autism spectrum disorder (ASD) is a neurodevelopmental disease with a strong heritability, but recent evidence suggests that epigenetic dysregulation may also contribute to the pathogenesis of ASD. Especially, increased methylation at the MECP2 promoter and decreased MECP2 expression were observed in the brains of ASD patients. However, the causative relationship of MECP2 promoter methylation and ASD has not been established. In this study, we achieved locus-specific methylation at the transcription start site (TSS) of Mecp2 in Neuro-2a cells and in mice, using nuclease-deactivated Cas9 (dCas9) fused with DNA methyltransferase catalytic domains, together with five locus-targeting sgRNAs. This locus-specific epigenetic modification led to a reduced Mecp2 expression and a series of behavioral alterations in mice, including reduced social interaction, increased grooming, enhanced anxiety/depression, and poor performance in memory tasks. We further found that specifically increasing the Mecp2 promoter methylation in the hippocampus was sufficient to induce most of the behavioral changes. Our finding therefore demonstrated for the first time the casual relationship between locus-specific DNA methylation and diseases symptoms in vivo, warranting potential therapeutic application of epigenetic editing.
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