The present invention provides methods of systems and methods of site specific methylation.

Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, detecting a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease polypeptides, CRISPR RNAs, trans-activating CRISPR RNAs, guide RNAs, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are CRISPR systems for binding a target sequence of interest, wherein the CRISPR system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.

The present disclosure provides compositions and methods relating to modified mini-nucleosome core proteins and/ or delivery of nucleic acids. In particular, the present disclosure includes, among other things, non-viral proteinaceous vehicles for delivery of nucleic acids. In various embodiments, non-viral proteinaceous vehicles provided herein include (a) a nucleic acid binding domain; (b) a targeting domain; (c) a nucleic acid release domain; and, optionally, (d) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, the proteinaceous vehicles include one or more modified residues.

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.

Embodiments herein include engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein (e.g., C2c1) that enhances binding of the of the CRISPR complex to the binding site and/ or alters editing preference as compared to wild type. Embodiments disclosed further include viral vectors for delivery of CRISPR-Cas effector proteins. The vectors may be designed to allow packaging of the CRISPR-Cas effector protein within a single vector. Certain embodiments further include delivery vectors, constructs, and methods of delivering larger genes for systemic delivery.

A method of treating a cardiomyopathy in a subject includes administering directly to or expressing locally in a weakened, ischemic, and/or peri-infarct region of myocardial tissue of the subject an amount of SDF-1 effective to cause functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test, or New York Heart Association (NYHA) functional classification.

Described in several exemplary embodiments are compositions including a targeting moiety effective to target a central nervous system cell and formulations thereof. In certain embodiments, the targeting moiety is composed of a n-mer motif, P motif, or both. Also described in certain example embodiments are vector systems configured to generate polypeptides containing the one or more targeting moieties. Also described herein are methods of generating a targeting moiety effective to target a central nervous system cell and using the compositions containing the targeting moieties described herein, such as to deliver a cargo to a subject and/or treat a central nervous system disease, disorder, or system thereof.

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.

The present invention encompasses engineered meganucleases which recognize and cleave a recognition sequence within the human PCSK9 gene. The present invention also encompasses methods for using such engineered meganucleases in a pharmaceutical composition and in methods for treating or reducing the symptoms of cholesterol-related disorders, such as hypercholesterolemia. Further, the invention encompasses pharmaceutical compositions comprising engineered meganuclease proteins, nucleic acids encoding engineered meganucleases, and the use of such compositions for treating cholesterol-related disorders, such as hypercholesterolemia.

Disclosed herein are viral vectors for use in recombinant molecular biology techniques. In particular, the present disclosure relates to self-limiting viral vectors containing nucleic acid sequences that encode engineered nucleases as well as nuclease recognition sequences such that expression of the engineered nuclease in a cell cleaves the viral vector and limits its persistence time. In some embodiments, the viral vectors disclosed herein also carry directives to delete, insert, or change a target sequence.