Disclosed are compositions and methods for gene editing. The present disclosure relates to compositions and methods for gene editing using a Cas nickase to cleave a double-stranded nucleic acid sequence near a target site and a ligase to incorporate a nucleic acid into a double-stranded nucleic acid sequence. The present disclosure also provides reagents for use in the gene editing methods. The present disclosure further provides kits containing reagents for use in the gene editing methods.

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.

Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.