DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine. 2020

Iván Devesa-Guerra, and Teresa Morales-Ruiz, and Juan Pérez-Roldán, and Jara Teresa Parrilla-Doblas, and Macarena Dorado-León, and María Victoria García-Ortiz, and Rafael R Ariza, and Teresa Roldán-Arjona
Maimónides Biomedical Research Institute of Córdoba (IMIBIC), 14071, Córdoba, Spain; Department of Genetics, University of Córdoba, 14071, Córdoba, Spain; Reina Sofía University Hospital, 14071, Córdoba, Spain.

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

UI MeSH Term Description Entries
D009687 Nuclear Proteins Proteins found in the nucleus of a cell. Do not confuse with NUCLEOPROTEINS which are proteins conjugated with nucleic acids, that are not necessarily present in the nucleus. Nucleolar Protein,Nucleolar Proteins,Nuclear Protein,Protein, Nuclear,Protein, Nucleolar,Proteins, Nuclear,Proteins, Nucleolar
D000072669 Gene Editing Genetic engineering or molecular biology techniques that involve DNA REPAIR mechanisms for incorporating site-specific modifications into a cell's genome. Base Editing,Genome Editing,Editing, Base,Editing, Gene,Editing, Genome
D000076987 CRISPR-Associated Protein 9 An RNA-guided endodeoxyribonuclease that associates with CRISPR SEQUENCES in STREPTOCOCCUS PYOGENES and other bacteria where it participates in an adaptive immune function to cleave foreign DNA complimentary to small GUIDE RNA (sgRNAs). Structurally, Cas9 consists of an ALPHA-HELIX module and a nuclease module connected by a single helix. The nuclease module contains two enzymatic domains: RuvC, which cleaves non-target DNA strand, and an HNH nuclease domain, which cleaves the target strand. Specificity for the DNA target depends on the presence of a protospacer adjacent motif (PAM) sequence, a 2-6 nucleotide DNA sequence immediately following the sequence targeted by Cas9. Cas9 Endonuclease,Cas9 Enzyme,Cas9 Protein,CRISPR Associated Protein 9,Endonuclease, Cas9,Enzyme, Cas9
D015533 Transcriptional Activation Processes that stimulate the GENETIC TRANSCRIPTION of a gene or set of genes. Gene Activation,Genetic Induction,Transactivation,Induction, Genetic,Trans-Activation, Genetic,Transcription Activation,Activation, Gene,Activation, Transcription,Activation, Transcriptional,Genetic Trans-Activation,Trans Activation, Genetic
D017360 Arabidopsis A plant genus of the family BRASSICACEAE that contains ARABIDOPSIS PROTEINS and MADS DOMAIN PROTEINS. The species A. thaliana is used for experiments in classical plant genetics as well as molecular genetic studies in plant physiology, biochemistry, and development. Arabidopsis thaliana,Cress, Mouse-ear,A. thaliana,A. thalianas,Arabidopses,Arabidopsis thalianas,Cress, Mouse ear,Cresses, Mouse-ear,Mouse-ear Cress,Mouse-ear Cresses,thaliana, A.,thaliana, Arabidopsis,thalianas, A.
D044127 Epigenesis, Genetic A genetic process by which the adult organism is realized via mechanisms that lead to the restriction in the possible fates of cells, eventually leading to their differentiated state. Mechanisms involved cause heritable changes to cells without changes to DNA sequence such as DNA METHYLATION; HISTONE modification; DNA REPLICATION TIMING; NUCLEOSOME positioning; and heterochromatization which result in selective gene expression or repression. Epigenetic Processes,Epigenetic Process,Epigenetics Processes,Genetic Epigenesis,Process, Epigenetic,Processes, Epigenetic,Processes, Epigenetics
D044503 5-Methylcytosine A methylated nucleotide base found in eukaryotic DNA. In ANIMALS, the DNA METHYLATION of CYTOSINE to form 5-methylcytosine is found primarily in the palindromic sequence CpG. In PLANTS, the methylated sequence is CpNpGp, where N can be any base. 5-Methylcytosine Monohydrochloride,5 Methylcytosine,5 Methylcytosine Monohydrochloride,Monohydrochloride, 5-Methylcytosine
D029681 Arabidopsis Proteins Proteins that originate from plants species belonging to the genus ARABIDOPSIS. The most intensely studied species of Arabidopsis, Arabidopsis thaliana, is commonly used in laboratory experiments. Arabidopsis thaliana Proteins,Thale Cress Proteins,Proteins, Arabidopsis thaliana,thaliana Proteins, Arabidopsis
D064113 CRISPR-Cas Systems Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS. CRISPR Cas Systems,CRISPR-Cas System,System, CRISPR-Cas,Systems, CRISPR-Cas

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