The invention relates to the field of nucleic acid synthesis or sequencing, more specifically to methods for ab-initio synthesis of nucleic acids, comprising contacting a nucleotide with a free 3′-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of an archaeal DNA primase or a functionally active fragment and/or variant thereof, thereby covalently binding said nucleoside triphosphate to the free 3′-hydroxyl group of the nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of both ab-initio single-stranded nucleic acid synthesis activity and template-independent terminal nucleotidyl transferase activity.

The invention relates to methods for template-independent synthesis of nucleic acids, comprising iteratively contacting an initiator sequence comprising a 3′-end nucleotide with a free 3′-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of an archaeal DNA primase or a functionally active fragment and/or variant thereof, thereby covalently binding said nucleoside triphosphate to the free 3-hydroxyl group of the 3-end nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of template-independent terminal nucleotidyl transferase activity but are devoid of a template-independent primase activity.

The invention is directed to compositions and methods for enzymatic template-free synthesis of polynucleotides wherein different terminal deoxynucleotidyl transferase (TdT) variants are used to incorporate different 3′-O-reversibly blocked deoxynucleoside triphosphates (dNTPs) into a growing chain. In part, the invention is a recognition and appreciation that different TdT variants can be engineered to preferentially incorporate specific dNTPs with higher efficiency than a single “general purpose” TdT variant used to incorporate all four 3-O-reversibly blocked dNTPs.

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides using chain elongation conditions that suppress the formation of DNA secondary structures including, but not limited to, intra-strand and between-strand duplexes, G-quadruplexes, and the like. In some embodiments, such chain elongation conditions include using 3′-O-blocked dNTP monomers that base protection groups or base analogs that suppress the formation of hydrogen bonding in the polynucleotide being synthesized.

The present invention is directed to methods and compositions for template-free enzymatic synthesis of a polyribonucleotide of a predetermined sequence from 3′-O-reversibly blocked nucleoside triphosphates using poly(A) and poly(U) polymerases.

The invention is directed to methods and apparatus for parallel enzymatic synthesis of polynucleotides in an array of reaction chambers using a tem-plate-free polymerase that has sequence-dependent coupling efficiencies. Whenever sequences causing low efficiency coupling occur at a 3′ end of a growing chain of a polynucleotide being synthesized, one or more additional coupling cy-cies without de-protection steps are inserted into synthesis plans to provide additional time for completing the coupling reaction at that position of the polynucleotide.

The invention relates to mutant DNA polymerases of the Pol theta subfamily capable of performing non-templated nucleic acid extension, or of a functional fragment of such a polymerase, methods of producing these mutant DNA polymerases, kits and methods of using these mutant DNA polymerases.

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).

The invention relates to improved methods of enzymatic solid-supported nucleic acid synthesis that make use of terminal deoxynucleotidyl transferase (TdT) enzymes or modified terminal deoxynucleotidyl transferase (TdT) enzymes on polyacrylamide type supports. The invention further relates to the use of kits comprising said enzymes in a method of solid-supported nucleic acid synthesis.