Abstract: Provided herein are compositions and methods for use of self-inactivating recombinant vectors (SIRV) encoding Class 2 Type V and guide ribonucleic acid (gRNA) sequences useful for nucleic acid sequence editing, and including self-inactivating components. The SIRV may be delivered to cells as part of an AAV vector to target a gene of interest.

Abstract: Provided herein are Class 2 Type V systems comprising nucleases, guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a C9orf72 gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations or duplications in the C9orf72 gene. Also provided are methods of using such systems to modify cells having such mutations or duplications.

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 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 polynucleotides configured for incorporation into recombinant adeno-associated virus (AAV) vectors. The polynucleotides encode for CRISPR proteins, gRNA, and ancillary components of AAV vectors useful in the modification of target nucleic acids. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the target nucleic acid of a gene. Also provided are methods of using such AAV vectors to modify cells having such mutations.

Abstract: Provided herein are systems comprising Class2, Type V CRISPR polypeptides, guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a PCSK9 gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the PCSK9 gene. Also provided are methods of using such CasX:gNA systems to modify cells having such mutations.

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 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 CasX:gNA systems, and compositions and methods relating thereto, the systems comprising CasX proteins, guide nucleic acids (gNAs), and optionally donor template nucleic acids useful for the modification cell genes encoding proteins involved in antigen processing, antigen presentation, antigen recognition, and/or antigen response, as well as methods of producing and using populations of cells comprising these modified genes. In some embodiments, the modified cells further express chimeric antigen receptors (CAR) or engineered T cell receptors (TCR). Such systems are useful for preparing cells for immunotherapy.

Abstract: Provided herein are CRISPR:guide systems comprising Class 2 Type V polypeptides (e.g. CasX:gNA systems comprising CasX polypeptides), guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a HTT gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the huntingtin 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 HTT-related disease, such as Huntington's disease.

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: 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: 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 CasX systems and components thereof, including variant CasX proteins and variant guide nucleic acids (gNAs). The variant CasX proteins and variant gNAs of the disclosure display at least one improved characteristic when compared to a reference CasX protein or reference gNA of the disclosure. In some instances, the variants have one or more improved CasX ribonucleoprotein complex functions. Also provided are methods of making and using said 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.