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 disclosure provides, in various embodiments, fusion proteins comprising a DNA-binding domain, a DNMT3A-binding domain, and a H3K4me0; and polynucleotides and vectors encoding one or more of the fusion proteins. The disclosure also provides, in various embodiments, gene-delivery systems, cells, compositions (e.g., pharmaceutical compositions) and kits comprising one or more of the fusion proteins polynucleotides, or vectors; methods of epigenetically modifying a genomic locus in a cell; and methods of treating a subject (e.g., a human) in need thereof.

The present disclosure relates to a method for increasing lipid content in microorganisms. The method comprises decreasing the expression of molecules involved in the protein synthesis to decrease protein synthesis and thereby increase lipid synthesis in the microorganisms. The present disclosure also provides a modified microorganism having increased lipid content.

The disclosure provides, in various embodiments, fusion proteins comprising a DNA-binding domain, a DNMT3A-binding domain, and a H3K4me0; and polynucleotides and vectors encoding one or more of the fusion proteins. The disclosure also provides, in various embodiments, gene-delivery systems, cells, compositions (e.g., pharmaceutical compositions) and kits comprising one or more of the fusion proteins polynucleotides, or vectors; methods of epigenetically modifying a genomic locus in a cell; and methods of treating a subject (e.g., a human) in need thereof.

The invention provides for a composition, which in particular comprising a fusion molecule comprising a least one DNA binding protein and at least one modulator of gene expression, wherein the fusion molecule is targeted to a genomic region near a VEGF gene and/or within a VEGF regulatory element and the at least one modulator of gene expression provides a modification of at least one nucleotide near the VEGF gene and/or within a VEGF regulatory element. The invention also provides methods of reduction or elimination of VEGF gene products in vivo and uses thereof.

The disclosure provides constructs comprising a first fusion protein, a second fusion protein, and a linker, wherein the first fusion protein and the second fusion protein each include an affinity reagent and a reactive enzyme, and the linker includes a first and second functional groups specific for irreversibly inhibiting the first and second fusion protein reactive enzymes. The disclosure further provides a method including (a) contacting a first fusion protein including an affinity reagent and a reactive enzyme with a linker including a functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme thereby coupling the first fusion protein and the linker, and (b) contacting a second fusion protein including an affinity reagent and a reactive enzyme with the linker, the linker including a functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme thereby coupling the second fusion protein and the linker.

Reduced genome bacteria with improved genetic stability are provided. Also provided are methods of producing polypeptides using the reduced genome bacteria with improved genetic stability.

Disclosed is a method for introducing an exogenous DNA by overcoming the restriction modification barrier of the target bacterium. The method includes the steps of 1) co-expressing all DNA-methyltransferase-encoding genes in the genome of the target bacterium in E. coli in which the restriction modification system thereof has been deleted to obtain a recombinant bacterium A, 2) introducing an exogenous DNA molecule into the recombinant bacterium A for in vivo modification so as to obtain a methylation-modified exogenous DNA molecule, and 3) introducing the methylation-modified exogenous DNA molecule into the target bacterium.

Methods, compositions and kits are provided to amplify an amount of genomic methylated DNA that can be 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.

Disclosed herein are artificially synthesized nucleic acid constructs to guide an epigenetic modification for at least partially silencing or activating a target gene in an organism such as a plant or seed, and formulations thereof. Also disclosed are methods of applying such nucleic acid constructs to the plant or to the seed. Also disclosed are engineered seeds and plants obtained by the epigenetic modification.