Provided herein are methods and agents for gene specific demethylation and/or activation. Oligonucleotide constructs are provided, the oligonucleotide constructs including: [1] a targeting portion having sequence complementarity and binding affinity with a region of genomic DNA within a gene, near a gene, or both; and [2] a single guide RNA (sgRNA) scaffold portion, wherein a tetra-loop portion of the sgRNA is modified and includes an R2 stem loop of DNMT1-interacting RNA (DiR), and wherein a stem loop 2 portion of the sgRNA is modified and includes an R5 step loop of DiR. The oligonucleotide constructs may be used, together with deactivated (dead) Cas9 (dCas9) for providing gene specific demethylation and/or activation of gene(s) of interest in a cell or subject in need thereof.

Examples provided herein are related to detecting methylcytosine and its derivatives using S-adenosyl-L-methionine analogs (xSAMs). Compositions and methods for performing such detection are disclosed. A target polynucleotide may include cytosine (C) and methylcytosine (mC). The method may include (a) protecting the C in the target polynucleotide from deamination; and (b) after step (a), deaminating the mC in the target polynucleotide to form thymine (T). Protecting the C from deamination may include adding a protective group to the 5 position of the C, e.g., using a methyltransferase enzyme that adds the first protective group from an xSAM.

The present disclosure provides technologies for modulating gene expression.

This disclosure relates to the genetic modification of DNMT3A gene in immune cells. In certain embodiments, the modified immune cells may be used in adoptive T cells therapies to enhance immune responses against cancer or chronic infections. In certain embodiments, the disclosure relates to deleting, changing, or inserting nucleotides within the DNMT3A gene in immune cells, e.g., human CD8 T cells, such that the DNMT3A gene product does not function for methylation. In certain embodiments, modification of the DNMT3A gene provides an improvement in antigen-specific T cells functions and/or an enhancement of the longevity of the cells.

Methods, compositions and kits are provided to amplify the amount of genomic methylated DNA can by subsequently analyzed and/or sequenced. It has particular use with small amounts of DNA, including, but not limited to cell free DNA samples. In some embodiments, the ratio of polymerase and methyltransferase is controlled in order to provide maximum yields. In some embodiments, a dual primase/polymerase is used.

Herein described is a construct for epigenomic modification of genes composed of: (a) a Krüppel-associated box zinc finger protein or homologous, (b) a DNA region capable of binding to the target gene or homologous, (c) a human DNA methyltransferase DNMT3A or homologous and (d) a murine DNA methyltransferase Dnmt3L or homologous, wherein components a), b), c) and d) are linked to each other either directly or via at least one linker. The construct is a designer epigenome modifier that can be used to silence genes coding for a protein in leukocytes that avoids the internalization of HI viruses in immune cells.

This disclosure relates to the genetic modification of DNMT3A gene in immune cells. In certain embodiments, the modified immune cells may be used in adoptive T cells therapies to enhance immune responses against cancer or chronic infections. In certain embodiments, the disclosure relates to deleting, changing, or inserting nucleotides within the DNMT3A gene in immune cells, e.g., human CD8 T cells, such that the DNMT3A gene product does not function for methylation. In certain embodiments, modification of the DNMT3A gene provides an improvement in antigen-specific T cells functions and/or an enhancement of the longevity of the cells.

The present invention relates to chimeric RNA oligonucleotides that are single-stranded oligonucleotides. These compounds are capable of targeting particular genes and reducing DNA methyltransferase activity. Accordingly, these compounds are particularly useful in the treatment of disease associated with aberrant DNA methyltransferase activity, such as cancer or a genetic disorder.

Methods for reactivating genes on the inactive X chromosome that include administering one or both of a DNA methyltransferase (DNMT) Inhibitor and/or a topoisomerase inhibitor, e.g., etoposide and/or 5-azacytidine (aza), optionally in combination with an inhibitor of XIST RNA and/or an Xist-interacting protein, e.g., a chromatin-modifying protein, e.g., a small molecule or an inhibitory nucleic acid (such as a small inhibitory RNA (siRNAs) or antisense oligonucleotide (ASO)) that targets XIST RNA and/or a gene encoding an Xist-interacting protein, e.g., a chromatin-modifying protein.

The invention relates to genetic engineering, and more particularly to a Dnmt3b gene-deficient CHO cell line, a preparation method and an application thereof and a recombinant protein expression system using the same. The invention adopts a CRISPR/Cas9 gene editing technique to knock out the Dnmt3b gene from the CHO cells to produce the Dnmt3b gene-deficient CHO cell line, which can significantly improve the expression level and stability of the target gene in CHO cells, overcoming the defects existing in the current CHO cell expression system, such as low expression level and stability. It has been demonstrated that using the CHO line provided herein to express a recombinant adalimumab can significantly increase the expression level of the recombinant adalimumab, indicating that the CHO cell line can be widely used to enhance the expression of target proteins.