Methods for CRISPR Enabled DNA Synthesis and compositions arising from the methods are provided. The methods may include ligation of partially single stranded DNA donor and acceptor oligonucleotides that are covalently linked to a subsequence of the target DNA to be sequenced followed by cleavage of the ligated product. In this manner the donor and acceptor oligonucleotides shuttle a growing subsequence of the target DNA with each cycle. A mutant Cpfl nuclease is missing non-specific ssDNA nuclease activity may be used for cleavage of the ligation product. Fourteen ligation/cleavage cycles can result in synthesis of ssDNA of greater than 10,000 bp in length.

Methods for CRISPR Enabled DNA Synthesis and compositions arising from the methods are provided. The methods may include ligation of partially single stranded DNA donor and acceptor oligonucleotides that are covalently linked to a subsequence of the target DNA to be sequenced followed by cleavage of the ligated product. In this manner the donor and acceptor oligonucleotides shuttle a growing subsequence of the target DNA with each cycle. A mutant Cpfl nuclease is missing non-specific ssDNA nuclease activity may be used for cleavage of the ligation product. Fourteen ligation/cleavage cycles can result in synthesis of ssDNA of greater than 10,000 bp in length.

The present disclosure is drawn to compositions, methods, and systems for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium. The composition can comprise one or more target primers, a DNA polymerase, and a re-solubilization agent. The composition can be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium. The method can comprise providing an assembly of a solid phase medium, depositing a biological sample onto the solid phase medium, and heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction. The system can comprise a composition and a solid phase medium on to which the composition is deposited.

The present disclosure is drawn to compositions, methods, and systems for loop-mediated isothermal amplification (LAMP) analysis on a solid phase medium. The composition can comprise one or more target primers, a DNA polymerase, and a re-solubilization agent. The composition can be substantially free of non-pH sensitive agents capable of discoloring the solid phase medium. The method can comprise providing an assembly of a solid phase medium, depositing a biological sample onto the solid phase medium, and heating the assembly to an isothermal temperature sufficient to facilitate a LAMP reaction. The system can comprise a composition and a solid phase medium on to which the composition is deposited.

The present application provides methods, compositions, and kits for analyzing a biological sample embedded in a three-dimensional polymerized matrix using a primer-exchange reaction (PER). In some embodiments, the methods comprise contacting the sample with a nucleic acid molecule that directly or indirectly binds to an analyte in the sample and immobilizing the nucleic acid molecule in the matrix, wherein the nucleic acid molecule comprises a free 3′ priming region for initiation of PER. In some embodiments, the methods enable sensitive detection of the identity and relative position of analytes in the sample.

The present application provides methods, compositions, and kits for analyzing a biological sample embedded in a three-dimensional polymerized matrix using a primer-exchange reaction (PER). In some embodiments, the methods comprise contacting the sample with a nucleic acid molecule that directly or indirectly binds to an analyte in the sample and immobilizing the nucleic acid molecule in the matrix, wherein the nucleic acid molecule comprises a free 3′ priming region for initiation of PER. In some embodiments, the methods enable sensitive detection of the identity and relative position of analytes in the sample.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

DNA is sequenced by: (a) combining dsDNA fragments with Y-adapters and hairpin adapters comprising an affinity-label under conditions wherein the adapters ligate to fragments forming a mixture of fragment inserts flanked by two Y-adapters, a Y-adapter and a hairpin adapter, and two hairpin adapters; and (b) sequencing the selected fragment inserts with sequencing primers selecting for the Y-adapters.

DNA is sequenced by: (a) combining dsDNA fragments with Y-adapters and hairpin adapters comprising an affinity-label under conditions wherein the adapters ligate to fragments forming a mixture of fragment inserts flanked by two Y-adapters, a Y-adapter and a hairpin adapter, and two hairpin adapters; and (b) sequencing the selected fragment inserts with sequencing primers selecting for the Y-adapters.