A Class 2, Type VI clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) which comprises a direct repeat (DR) stem loop sequence and a guide or spacer sequence, is provided characterized by a DR selected from those of Table 9. Also described is are methods for generating, selecting, characterizing and optimizing a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) for use in the CRISPR-Cas13d system described herein. Also provided is a screening method to identify crRNA particularly suited for use with specified targets. Further, the invention includes non-naturally occurring, synthesized or engineered crRNAs as described herein along with nucleic acid molecule, vectors, RNPs, cells, libraries, and compositions comprising the same, and uses thereof in treating a disease or in functionally screening a gene. A method for blocking an RNA target without degradation and a method for modification of multiple RNA targets using the same CRISPR effector protein are also disclosed.

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

The present invention provides a composition comprising dendritic cells loaded with hHsp60sp, which dendritic cells are from a subject and have been fixed with paraformaldehyde (PFA). The subject may suffer from an autoimmune disease. Also provided are a method for preparing the composition; recombinant human cells comprising a heterologous gene encoding a fusion protein of HLA-E and hHsp60sp or B7sp, and expressing the fusion protein on the surface of the cells; a method for determining a percentage of maximum inhibition of testing the function of the HLA-E restricted CD8+ Treg cells from a subject, determining whether HLA-E restricted CD8+ Treg cells freshly isolated from a subject are defective, or determining whether defective HLA-E restricted CD8+ Treg cells from a subject are correctable; and a method for correcting defective HLA-E restricted CD8+ Treg cells, treating type 1 diabetes (T1D), or treating multiple sclerosis (MS).

The invention relates to methods for the simultaneous expression and delivery to mitochondria of two or more proteins using a single expression vector. Also described are the expression vectors and host cells comprising the vectors. Where the proteins are genome editing reagents, the invention also relates to the use of the expression vectors to alter levels of mitochondrial heteroplasmy and treat mitochondrial disorders.

The invention provides recombinant DNA molecules useful for providing efficient expression of proteins in transgenic plants, as well as compositions and methods for using the recombinant DNA molecules. In particular embodiments, the invention provides recombinant DNA molecules and constructs comprising sequences encoding transit peptides and operably linked sequences conferring herbicide tolerance.

The present disclosure provides improved adenosine base editors (ABE) that have an expanded range of PAM sequence recognition capability (i.e., recognition of non-canonical ′5-NGG-′3 PAM sequence). In addition, the present disclosure provides improved cytidine base editors (CBE) and adenosine base editors (ABE) comprising circular permutant variants of Cas9 (CP-Cas9) with an increased window of base editing within the protospacer sequence (e.g., from about 4-5 nucleotides to up to about 8-9 nucleotides) and even outside of the protospacer sequence.

Provided herein are compositions that include proteins that include a mitochondrial localization amino acid sequence attached to a base editor fusion protein including a nucleotide base-modifying enzyme and an RNA-guided DNA endonuclease enzyme with modified endonuclease activity. Provided herein are nucleic acids encoding the proteins and vectors including the nucleic acids. Provided herein are pharmaceutical compositions including the compositions, proteins, nucleic acid and vectors. Provided herein are related methods for modifying mitochondrial DNA and treating mitochondrial disorders.

Parkin protein variants having activating mutations and/or fused to a mitochondrial targeting sequence are provided. The engineered Parkin may be a fusion protein including a mitochondrial targeting sequence (MTS); a transmembrane domain; and a Parkin protein or functional variant or fragment thereof, such as a Parkin having an N-terminal deletion. The MTS may be the MTS of PINK1 or a functional variant thereof. Alternatively or in addition, the engineered Parkin may have one or more activating mutations, such as single amino-acid substitutions. The engineered Parkin may be delivered in a vector, such as an adeno-associated virus (AAV) vector, and may be used to treat a disease or disorder, such as Parkinson’s disease or any of various neurodegenerative diseases.

The present invention provides: a complex comprising a genome editing enzyme and a cell membrane-permeable peptide(CPP), wherein the CPP is fused to the genome editing enzyme; a complex comprising a genome editing enzyme, a target gene-specific nucleic acid, and a CPP, wherein the CPP is fused to the genome editing enzyme and/or the a target gene-specific nucleic acid; the complex comprising a polycationic moiety fused to the CPP, wherein the polycation moiety is statically bound to the target gene-specific nucleic acid; a genome editing method using the complex; and a kit for genome-editing, the kit including these complexes.