Engineered nucleases (e.g., zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and others) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 10.sup.11 DNA sequences for their ability to be cleaved by active, dimeric nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future nuclease design. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

Engineered nucleases are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. This disclosure provides in vitro selection methods that interrogate 10.sup.11 DNA sequences for their ability to be cleaved by active nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci. This disclosure provides an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

Engineered nucleases are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. This disclosure provides in vitro selection methods that interrogate 10.sup.11 DNA sequences for their ability to be cleaved by active nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci. This disclosure provides an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

Automated methods conducted in a sequencing flowcell, and kits for reusing a flowcell, are provided herein. In some examples, an automated method conducted in a sequencing flowcell may include, at a surface of the sequencing flowcell coupled to a first moiety, using a reagent to decouple a first complex from the first moiety. In some examples, the first complex may include a second moiety which couples to the first moiety and a polynucleotide coupled to the second moiety. In some examples, the method may further include using a nuclease to polynucleotides in the sequencing flowcell. The method may include, after using the reagent and after using the nuclease, coupling a second complex to the first moiety. The second complex may include a third moiety which couples with the first moiety and an oligonucleotide coupled to the third moiety.

Automated methods conducted in a sequencing flowcell, and kits for reusing a flowcell, are provided herein. In some examples, an automated method conducted in a sequencing flowcell may include, at a surface of the sequencing flowcell coupled to a first moiety, using a reagent to decouple a first complex from the first moiety. In some examples, the first complex may include a second moiety which couples to the first moiety and a polynucleotide coupled to the second moiety. In some examples, the method may further include using a nuclease to polynucleotides in the sequencing flowcell. The method may include, after using the reagent and after using the nuclease, coupling a second complex to the first moiety. The second complex may include a third moiety which couples with the first moiety and an oligonucleotide coupled to the third moiety.

In certain embodiments, the present invention provides a detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide. As used herein, the term picodroplet comprises a liquid droplet that has a volume of 0.014 to 2.6 picoliters. In certain embodiments, the present invention provides a method of detecting at least one individual nuclease molecule present in a sample, comprising contacting an aqueous sample suspected of containing at least one nuclease with at least one detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide to form an aqueous reaction mixture; emulsifying the aqueous mixture in oil to form picoliter-scale droplets in an emulsion, (c) incubating the picoliter-scale droplets in the emulsion in order for the nuclease, if present, to digest the substrate probes linked to the microbeads; recovering the microbeads; and detecting fluorescence emitting from the microbeads.

In certain embodiments, the present invention provides a detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide. As used herein, the term picodroplet comprises a liquid droplet that has a volume of 0.014 to 2.6 picoliters. In certain embodiments, the present invention provides a method of detecting at least one individual nuclease molecule present in a sample, comprising contacting an aqueous sample suspected of containing at least one nuclease with at least one detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide to form an aqueous reaction mixture; emulsifying the aqueous mixture in oil to form picoliter-scale droplets in an emulsion, (c) incubating the picoliter-scale droplets in the emulsion in order for the nuclease, if present, to digest the substrate probes linked to the microbeads; recovering the microbeads; and detecting fluorescence emitting from the microbeads.

Methods and compositions are described herein that are useful for analyzing and identifying retron systems with improved replication fidelity and efficiency. The methods and compositions described herein facilitate the engineering, quantification, and identification of retron elements for precise genome engineering.

Methods and compositions are described herein that are useful for analyzing and identifying retron systems with improved replication fidelity and efficiency. The methods and compositions described herein facilitate the engineering, quantification, and identification of retron elements for precise genome engineering.

Disclosed are methods, compositions and kits for contacting a sample containing a biological particle with a catalyst associated with or attached to a support. The biological particle may be cells and/or nuclei. The catalyst may be an enzyme configured to digest an extracellular molecule, such as an extracellular biological molecule, including extracellular nucleic acid molecules. In some examples, the biological particle is an aggregate of cells that is processed to single cells with a nuclease that is attached to a bead support. The bead and nuclease may subsequently be removed from the system. The single cells that result from the method can be used in single cell-based droplet systems for obtaining genome or transcriptome profiles of single cells.