The present invention relates to engineered Clustered Regularly Interspaced Short Palindromic Repeals (CRISPR) systems and corresponding guide RNAs that target specific nucleotide sequences at certain gene loci in the human genome. Also provided are methods of targeting, editing, and/or modifying of the human genes using the engineered CRISPR systems, and compositions and cells comprising the engineered CRISPR systems.

Compositions and methods for modifying genomic DNA sequences are provided. The methods produce double-stranded breaks (DSBs) at pre-determined target sites in a targeted DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the targeted site(s). Compositions comprise DNA constructs comprising nucleotide sequences that encode a Cpf1 protein operably linked to a promoter that is operable in the cells of interest. The DNA constructs can be used to direct the modification of genomic DNA at pre-determined locations. Methods to use these DNA constructs to modify genomic DNA sequences are described herein. Additionally, compositions and methods for modulating the expression of genes are provided. Compositions comprise DNA constructs comprising a promoter that is operable in the cells of interest operably linked to nucleotide sequences that encode a mutated Cpf1 protein with an abolished ability to produce DSBs, optionally linked to a domain that regulates transcriptional activity. The methods can be used to up- or down-regulate the expression of genes at predetermined genomic loci.

The present disclosure provides D1L3 enzymes having complete or partial C-terminal deletions of the basic domain (BD), which have substantially enhanced chromatin-degrading activity. In accordance with aspects of the invention, the D1L3 enzymes described herein are more suitable and/or effective for therapy and/or are more amenable to large-scale manufacturing. In some embodiments, the D1L3 enzymes have benefits for systemic therapy. Such benefits include longer exposure (e.g., slower elimination, longer circulatory half-life), extended duration of pharmacodynamic action, and improved chromatin-degrading activity.

The present invention relates to an artificially engineered CRISPR/Cas9 system. More particularly, the present invention relates to an artificially engineered CRISPR enzyme having enhanced target specificity and a use of an artificially engineered CRISPR/Cas9 system including the same enzyme in genome and/or epigenome manipulation or modification, genome targeting, genome editing, and in vitro diagnosis, etc.

The present invention features compositions and methods for editing deleterious mutations associated with hemoglobinopathies, such as sickle cell disease (SCD). In particular embodiments, the invention provides methods for correcting mutations in a beta globin polynucleotide using modified adenosine base editors termed “ABE8” having unprecedented levels (e.g., >60-70%) of efficiency.

The disclosure provides adenosine deaminases that are capable of deaminating adenosine in DNA to treat cancers, such as melanoma and glioblastoma. The disclosure also provides fusion proteins, guide RNAs and compositions comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA, for example in a STAT3 gene. In some embodiments, adenosine deaminases provided herein are used to modify the STAT3 gene so that its protein product, STAT3, is unable to be activated. In some embodiments, the methods and compositions provided herein are used to treat melanoma or glioblastoma.

Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor and/or survival factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes a survival factor, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor and/or a different survival factor.

Polynucleotides comprising the following base sequences: (a) a base sequence encoding a fusion protein of a nuclease-deficient CRISPR effector protein and a transcription activator, and (b) a base sequence encoding a guide RNA targeting a continuous region of 18 to 24 nucleotides in length in a region set forth in SEQ ID NO: 104, 105, 135, 141, 153, 167, or 172 in the expression regulatory region of human Utrophin gene are expected to be useful for treating or preventing DUCHENNE muscular dystrophy or BECKER muscular dystrophy.

The present invention provides chimeric Flp-TAL recombinases, as well as nucleic acids, and methods for the use of the chimeric Flp-TAL recombinases for site-specific alteration of a target sequence in cells.

Aspects of the disclosure relate to compositions and methods for exon replacement in a cell or a subject. In some embodiments, the disclosure relates to isolated nucleic acids (and vectors, such as rAAV vectors) encoding one or more guideRNAs (gRNAs) that target an intron-exon boundary; an intronic sequence having a splice signal; and a donor sequence encoding a gene product of a gene of interest, or portion thereof. In some embodiments, compositions described herein are useful for replacing mutant exons associated with certain diseases, for example Duchen's muscular dystrophy (DMD), cystic fibrosis (CF), spinal muscular atrophy (SMA), Rett syndrome, and mucopolysaccharidosis (MPS).