Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.

The disclosure relates to compositions comprising and methods for chemical modification of single guide RNA (sgRNA), tracrRNA and/or crRNA used individually or in combination with one another or Cas system components. Compositions comprising modified ribonucleic acids have been designed with chemical modification for even higher efficiency as unmodified native strand of sgRNA. Administration of modified ribonucleic acids will allow decreased immune response when administered to a subject, increased stability, increased editing efficiency and facilitated in vivo delivery of sgRNA via various delivery platforms. The disclosure also relates to methods of decreasing off-target effect of CRISPR and a CRISPR complex.

Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.

Provided herein are compositions for and methods of generating ligation-competent nucleic acids. In some aspects, the compositions comprise Exonuclease III, T4 DNA Polymerase, Klenow, and/or T4 polynucleotide kinase.

A composition used in targeted mutagenesis is provided, which includes a first expression cassette comprising a nucleotide sequence which encodes a CAS9 endonuclease; a second expression cassette comprising a nucleotide sequence which encodes a guide RNA sequence, wherein the guide RNA sequence is complementary to a target genome nucleotide sequence in a cell; and a third expression cassette comprising a nucleotide sequence which encodes a Trex2 exonuclease (Trex2) gene. The first, second, and third expression cassettes may be a part or a portion of one or more expression vectors.

This disclosure relates to methods and compositions for detecting a target nucleic acid sequence using a dual-hapten probe. More specifically, the present disclosure relates to using recombinase polymerase amplification (RPA) and a dual-hapten probe to detect a target nucleic acid sequence. In some cases, the detection is on lateral flow strips.

A composition used in targeted mutagenesis is provided, which includes a first expression cassette comprising a nucleotide sequence which encodes a CAS9 endonuclease; a second expression cassette comprising a nucleotide sequence which encodes a guide RNA sequence, wherein the guide RNA sequence is complementary to a target genome nucleotide sequence in a cell; and a third expression cassette comprising a nucleotide sequence which encodes a Trex2 exonuclease (Trex2) gene. The first, second, and third expression cassettes may be a part or a portion of one or more expression vectors.

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.

The present disclosure is directed to methods of producing a modified nucleic acid comprising a precise deletion in a target nucleic acid in a cell comprising generating, within the cell, a first single strand break on a first strand of the target nucleic acid and a second single strand break on a second strand of the target nucleic acid, thereby forming a double strand break in the target nucleic acid having a first 3 overhang and a second 3 overhang; processing the first 3 overhang and the second 3 overhang with an exonuclease molecule, thereby deleting the segment of the target nucleic acid that was located between the first single strand break and the second single strand break, and forming a processed double strand break; and allowing the processed double strand break to be repaired by at least one DNA repair pathway, thereby producing the modified nucleic acid comprising the precise deletion in the target nucleic acid in the cell. Gene editing systems, vectors, polynucleotides, and methods of treatment are also disclosed herein.

The present disclosure provides improved genome editing compositions and methods for editing a double-strand DNA target site. The disclosure further provides genome edited cells produced by the compositions and methods described.