Abstract: Provided herein are engineered Class 2, Type V nucleases and guide RNAs useful for the editing of target nucleic acids. Also provided are methods of making and using such variants to modify nucleic acids.

Abstract: Provided herein are reference guide nucleic acid scaffolds and variants of reference guide nucleic acid scaffolds capable of binding one or more engineered proteins comprising a RuvC cleavage domain. In some embodiments, the variants of the reference guide nucleic acid scaffolds comprise at least one modification compared to the reference guide nucleic acid scaffold sequences and exhibit one or more improved characteristics compared to the reference guide nucleic acid scaffolds.

Abstract: Provided herein are delivery particle systems (XDP) useful for the delivery of payloads of any type. In some embodiments, a XDP particle system with tropism for target cells of interest is used to deliver CRISPR/Cas polypeptides (e.g., CasX proteins) and guide nucleic acids (gNA), for the modification of nucleic acids in target cells. Also provided are methods of making and using such XDP to modify the nucleic acids in such cells.

Abstract: Provided herein are CasX:gNA systems comprising CasX polypeptides, guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a SOD1 gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the SOD1 protein or the SOD1 regulatory element. Also provided are methods of using such CasX:gNA systems to modify cells having such mutations and utility in methods of treatment of a subject with a SOD1-related disease.

Abstract: Provided herein are engineered proteins comprising a RuvC DNA cleavage domain comprising one or more amino acid modifications, and one or more improved characteristics, relative to a naturally occurring RuvC domain. Also provided are gene editing systems comprising engineered proteins, and methods for use thereof.

Abstract: Provided herein are Class 2 Type V CRISPR:gNA systems comprising Class 2 Type V CRISPR polypeptides (e.g. CasX), guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a RHO gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the rhodopsin protein. Also provided are methods of using such systems to modify cells having such mutations and utility in methods of treatment of a subject with a RHO-related disease, such as retinitis pigmentosa.

Abstract: The present disclosure provides variant Cas9 polypeptides, where a variant Cas9 polypeptide of the present disclosure comprises an internal insertion of a heterologous polypeptide. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the variant Cas9 polypeptides. The present disclosure provides host cells comprising a variant Cas9 polypeptide of the present disclosure, or comprising a nucleic acid encoding a variant Cas9 polypeptide of the present disclosure. The present disclosure provides methods of binding and/or modifying a target nucleic acid, involving use of a variant Cas9 polypeptide of the present disclosure.

Abstract: The present disclosure provides variant Cas9 polypeptides, where a variant Cas9 polypeptide of the present disclosure comprises an internal insertion of a heterologous polypeptide. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the variant Cas9 polypeptides. The present disclosure provides host cells comprising a variant Cas9 polypeptide of the present disclosure, or comprising a nucleic acid encoding a variant Cas9 polypeptide of the present disclosure. The present disclosure provides methods of binding and/or modifying a target nucleic acid, involving use of a variant Cas9 polypeptide of the present disclosure.

Abstract: The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a proto spacer adjacent motif (PAM) sequence and at least one of: a specificity segment positioned 5? of the PAM sequence, and an orientation segment positioned 3? of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.

Abstract: The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a protospacer adjacent motif (PAM) sequence and at least one of: a specifity segment positioned 5? of the PAM sequence, and an orientation segment positioned 3? of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.

Abstract: The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a proto spacer adjacent motif (PAM) sequence and at least one of: a specifity segment positioned 5? of the PAM sequence, and an orientation segment positioned 3? of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.

Abstract: The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a protospacer adjacent motif (PAM) sequence and at least one of: a specifity segment positioned 5? of the PAM sequence, and an orientation segment positioned 3? of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.

Abstract: The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a protospacer adjacent motif (PAM) sequence and at least one of: a specificity segment positioned 5? of the PAM sequence, and an orientation segment positioned 3? of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.