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 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 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 is directed to compositions and methods for enzymatic template-free synthesis of polynucleotides using terminal deoxynucleotidyl transferases that are chimeras derived from variants from different species.

The present invention is directed to terminal deoxynucleotidyl transferase (TdT) variants from a variety of species which display enhanced efficiency in incorporating reversibly blocked nucleoside triphosphates into a polynucleotide, and to the use of such TdTs in synthesizing polynucleotides of any predetermined sequence.

Provided is a method including extending a ssDNA by sequentially adding a plurality of modified nucleoside triphosphates to the ssDNA, wherein the base of the modified nucleoside triphosphates includes a primary modification selected from (i) a primary polynucleotide attached to the base of the modified nucleoside triphosphate, and (ii) a site on the base for covalent attachment of a primary polynucleotide to the base, further comprising covalently attaching a primary polynucleotide to the base after the polymerizing.