The present invention is directed to terminal deoxynucleotidyl transferase (TdT) variants from a variety of species which have been engineered for increase compactness and enhanced efficiency in incorporating reversibly blocked nucleoside triphosphates into a polynucleotide, especially polynucleotides attached porous solid supports. The invention includes use of such TdTs for synthesizing polynucleotides of any predetermined sequence.

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis.

Provided in some aspects are methods for light-controlled in situ surface patterning of a substrate comprising a step of blocking or ablating oligonucleotide molecules in a boundary region separating a plurality of spot regions, and attaching oligonucleotides to oligonucleotide molecules in a first one or more of the spot regions.

An enzymatic method of making a polynucleotide is provided. The method includes combining a selected nucleotide triphosphate, one or more cations, a template-independent polymerase, and an associated processivity factor in an aqueous reaction medium including a target substrate comprising an initiator sequence and having a 3 terminal nucleotide attached to a single stranded portion, such that the template-independent polymerase and the associated processivity factor interact with the target substrate under conditions which covalently add one or more of the selected nucleotide triphosphate to the 3 terminal nucleotide. Also provided are mutant template-independent polymerases having a processivity factor attached thereto.

The application provides improved methods of genome editing. The genome editing systems described herein comprise a RNA-guided nuclease molecule and a Repair-Modulating Enzyme Molecule (RMEM).

This application provides improved methods of genome editing. The genome editing systems described herein comprise a RNA-guided nuclease molecule and a Repair-Modulating Enzyme Molecule (RMEM).

The invention relates to variants of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand, or of a functional fragment of such a polymerase, comprising at least one mutation of a residue in at least one specific position, and to uses of said variants, in particular for the synthesis of nucleic acid molecules comprising 3-OH modified nucleotides.

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to a variant of Terminal deoxynucleotidyl Transferase (TdT) which (i) comprises the amino acid sequence as set forth in SEQ ID N 2 or a functionally equivalent sequence, with at least an amino acid substitution at position corresponding to residue C302 or functionally equivalent residue, wherein the position is numbered by reference to the amino acid sequence set forth in SEQ ID N 1, (ii) is able to synthesize a nucleic acid fragment without template and (iii) is able to incorporate a modified nucleotide into the nucleic fragment.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.