Abstract: Described herein are compositions, vectors, cells, methods, and kits that provide cell data recorder systems for recording cell states. The cell data recorder systems allow for the recording of both the presence and duration of one or more stimuli in a programmable, reproducible, and multiplexable manner. These cell data recorder systems employ a nucleic acid programmable DNA binding protein, such as a Cas9 nuclease, or a fusion protein comprising a nucleic acid programmable DNA binding domain and a nucleic acid editing domain to introduce recordable changes in the genome of a cell or in a plasmid within the cell.

Abstract: Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

Abstract: Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids or the modification of nucleic acids or proteins, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of nucleic acid programmable DNA binding proteins e.g., GeoCas9 or variants thereof, and effector domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing or protein modification are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a GeoCas9 and effector domains, are provided.

Abstract: Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

Abstract: Aspects of the disclosure relate to compositions and methods for targeted protein degradation. In some embodiments, the disclosure relates to methods of evolving protein degrons to interact with certain small molecule inducers (e.g., VS-777, PT-179, or PK-1016). In some embodiments, the disclosure relates to compositions (e.g., peptides, nucleic acids encoding the protein degrons, etc.) used for targeted protein degradation. In some embodiments, the disclosure relates to methods of degrading a target polypeptide in a cell.

Abstract: Provided herein are compounds of Formula (I-A), (I-B), or (I-C), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically enriched forms, prodrugs, or mixtures thereof, and compositions thereof. Also provided are methods and kits involving the inventive compounds or compositions for treating and/or preventing diseases and/or conditions (e.g., neurological disease (e.g., Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis), metabolic disorder (e.g., obesity, diabetes, X-linked adrenoleukodystrophy (X-ALD)), proliferative disease (e.g., cancers), hepatic disease (e.g., liver cirrhosis), conditions associated with autophagy (e.g., neurodegenerative disease, infection, cancer, conditions associated with aging, heart disease), conditions associated with aging, conditions associated with modulating the mPTP, cardiovascular conditions (e.g.

Abstract: The present disclosure provides Cas protein variants comprising one or more amino acid substitutions relative to wild-type Cas14a1. Fusion proteins comprising the Cas protein variants described herein are also provided by the present disclosure. Further provided herein are methods for modifying a target nucleic acid using the Cas proteins and fusion proteins provided herein. The present disclosure also provides guide RNAs, complexes, polynucleotides, systems, cells, kits, and pharmaceutical compositions.

Abstract: The disclosure provides adenosine deaminases that are capable of deaminating adenosine in DNA. The disclosure also provides fusion proteins comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA. In some embodiments, the fusion proteins further comprise a nuclear localization sequence (NLS), and/or an inhibitor of base repair, such as, a nuclease dead inosine specific nuclease (dISN).

Abstract: Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for engineering Cas9 and Cas9 variants that have increased activity on target sequences that do not contain the canonical PAM sequence. In some embodiments, fusion proteins comprising such Cas9 variants and nucleic acid editing domains, e.g., deaminase domains, are provided.

Abstract: The present disclosure provides zinc finger domain-containing proteins comprising optimized ?-, ?-, and linker motifs, and fusion proteins comprising said zinc finger domain-containing proteins fused to an effector domain. The present disclosure also provides double-stranded DNA deaminase A (DddA) variants and fusion proteins comprising said DddA variants fused to a programmable DNA binding protein (e.g., any of the zinc finger domain-containing proteins disclosed herein, a TALE protein, or a CRISPR/Cas9 protein). Methods for editing DNA (including genomic DNA and mitochondrial DNA) using the fusion proteins described herein are also provided by the present disclosure. The present disclosure further provides polynucleotides, vectors, cells, kits, and pharmaceutical compositions comprising the zinc finger domain-containing proteins, DddA variants, and fusion proteins described herein.

Abstract: Disclosed herein are compositions, methods, kits, and systems relating to efficient delivery of cargos (e.g., therapeutic cargos) into cells, for instance, for in vivo delivery. The present disclosure provides lipid-containing particles (e.g., virus-like particles) for delivering therapeutic cargos. The present disclosure also provides polynucleotides encoding the lipid-containing particles provided herein, which may be useful for producing said lipid-containing particles. Also provided are methods for editing nucleic acid molecules in cells using the lipid-containing particles provided herein, as well as cells and kits comprising the lipid-containing particles.

Abstract: Disclosed herein are compositions, methods, kits, and systems relating to efficient delivery of cargos (e.g., therapeutic cargos) into cells, for instance, for in vivo delivery. The present disclosure provides lipid-containing particles (e.g., virus-like particles) for delivering therapeutic cargos. The present disclosure also provides polynucleotides encoding the lipid-containing particles provided herein, which may be useful for producing said lipid-containing particles. Also provided are methods for editing nucleic acid molecules in cells using the lipid-containing particles provided herein, as well as cells and kits comprising the lipid-containing particles.

Abstract: Nucleic acid molecules, compositions, recombinant AAV (rAAV) particles, kits, and methods are described herein for delivering a base editor (or “nucleobase editor”) to cells, e.g., via AAV vectors. In particular, the disclosure provides compositions, methods, and uses for delivery of adenine base editors and cytosine base editors in a single AAV vector (or genome). Further described herein are improved AAV vectors containing size-minimized regulatory components that enable, e.g., the packaging of base editors. Provided herein are methods and compositions for delivering base editor proteins to a cell or tissue in a single recombinant AAV (rAAV) vector. Contemplated herein are improved methods and compositions for delivering these base editors in vivo, in a single rAAV particle. Further provided herein are base editors and compositions and cells comprising these base editors.

Abstract: The present disclosure provides virus-like particles (VLPs) for delivering prime editors, and systems comprising such prime editor (PE) VLPs. The present disclosure also provides polynucleotides encoding the PE-VLPs described herein, which may be useful for producing said PE-VLPs. Also provided herein are methods for editing the genome of a target cell by introducing the presently described PE-VLPs into the target cell. The present disclosure also provides fusion proteins that make up a component of the PE-VLPs described herein, as well as polynucleotides, vectors, cells, and kits.

Abstract: Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. The disclosure provides fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cas9 or variants thereof, and nucleic acid editing proteins such as cytidine deaminase domains (e.g., novel cytidine deaminases generated by ancestral sequence reconstruction), and adenosine deaminases that deaminate adenine in DNA. Aspects of the disclosure relate to fusion proteins (e.g., base editors) that have improved expression and/or localize efficiently to the nucleus. In some embodiments, base editors are codon optimized for expression in mammalian cells. In some embodiments, base editors include multiple nuclear localization sequences (e.g., bipartite NLSs), e.g., at least two NLSs.

Abstract: Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Abstract: Disclosed herein are compositions, methods, kits, and systems relating to efficient delivery of cargos (e.g., therapeutic cargos) into cells, for instance, for in vivo delivery. The present disclosure provides lipid-containing particles (e.g., virus-like particles) for delivering therapeutic cargos. The present disclosure also provides polynucleotides encoding the lipid-containing particles provided herein, which may be useful for producing said lipid-containing particles. Also provided are methods for editing nucleic acid molecules in cells using the lipid-containing particles provided herein, as well as cells and kits comprising the lipid-containing particles.

Abstract: The present disclosure provides Cas9 variants, and base editors comprising these variants, that recognize non-canonical protospacer adjacent motifs (PAMs) and have less restrictive PAM requirements for editing. The present disclosure provides Cas9 protein variants comprising one or more amino acid substitutions relative to wild-type Nme2Cas9. Fusion proteins comprising the Cas protein variants described herein are also provided by the present disclosure. Further provided herein are methods for editing a target nucleic acid using the Cas variants and fusion proteins provided herein. The present disclosure also provides guide RNAs, complexes, polynucleotides, cells, kits, and pharmaceutical compositions. Further described herein are phage-assisted continuous evolution (PACE) systems, vectors, methods, and devices.

Abstract: The specification provides programmable base editors that are capable of introducing a nucleotide change and/or which could alter or modify the nucleotide sequence at a target site in mitochondrial DNA (mtDNA) with high specificity and efficiency. Moreover, the disclosure provides fusion proteins and compositions comprising a programmable DNA binding protein (e.g., a mitoTALE, a mitoZFP, or a CRISPR/Casp) and double-stranded DNA deaminase that is capable of being delivered to the mitochondria and carrying out precise installation of nucleotide changes in the mtDNA. The fusion proteins and compositions are not limited for use with mtDNA, but also may be used for base editing of any double-stranded target DNA.

Abstract: The present disclosure provides new prime editor guide RNAs for prime editing, constructs for prime editing, and methods for using same. In addition, the present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis (e.g., insertion or deletion). The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a prime editor RNA (PEgRNA).