Abstract: Provided herein are methods of developing biomolecule variants (such as proteins, RNA, or DNA) with improved characteristics, for example by developing libraries of variants with alterations to one or more specific monomer locations and screening said libraries for characteristics of interest. These alterations can include deletion, substitution, and insertion, and variants may comprise one alteration or a combination of alterations. Said methods may include further iterative cycles of library construction and evaluation to develop, for example, a biomolecule variant with improved characteristics compared to a reference biomolecule. The methods can also provide information that may be used in the rational design of variants.

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. In some embodiments, the engineered proteins comprise a RuvC DNA cleavage domain comprising one or more modifications, relative to a naturally occurring domain. In some embodiments, the engineered proteins comprise a chimeric domain, for example a chimeric helical I domain. In some embodiments, the engineered proteins are chimeric proteins, comprising at least two domains, each domain derived from a different source, for example derived from SEQ ID NO: 1 and SEQ ID NO: 2. Also provided are systems comprising these engineered proteins, and methods of making and using said engineered proteins.

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.