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: Disclosed are constructs, systems, and methodologies using prime editing (PE), twin prime editing (twinPE), or multi-flap prime editing to carry out site-specific and large-scale genetic modification, such as, but not limited to, insertions, deletions, inversions, replacements, and chromosomal translocations of whole or partial genes (e.g., whole gene, gene exons and/or introns, and gene regulatory regions). In certain embodiments, the disclosure provides constructs, systems, and methods using prime editing (PE), e.g., single flap or “classical” PE or twinPE or multi-flap PE, to install one or more target sites for site specific recombination in a target genomic locus (e.g., a specific gene, exon, intron, or regulatory sequence), which may then be acted on by one or more site-specific recombinases to effectuate a large-scale genetic modification, such as an insertions, deletions, inversions, replacements, and chromosomal translocations.

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).

Abstract: The present disclosure provides compositions and methods for the targeted modification of RNA molecules by RNA prime editing. The compositions and methods may be conducted invitro or in vivo within cells (e.g., human cells) for the therapeutic correction of disease-causing mutations and/or installation of motifs or mutations in RNA molecules of interest as a tool for scientific research. The disclosure provides compositions and methods for conducting RNA prime editing of a target RNA molecule (e.g., an RNA transcript) that enables the incorporation of one or more nucleotide changes and/or targeted mutagenesis of a target RNA molecule. The nucleotide change can include a single-nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides.

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 a double-stranded nucleotide sequence, such as, a chromosome, genome, or a 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/Cas9) and evolved double-stranded DNA deaminase domains that is capable of being delivered to a cell nucleus and/or a mitochondria and carrying out precise installation of nucleotide changes in the target a double-stranded nucleotide sequence, such as, a chromosome, genome, or mtDNA. The fusion proteins and compositions are not limited for use with mtDNA, but may be used for base editing of any double-stranded target DNA.

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: The present disclosure provides systems, methods, and compositions for modifying the crumbs homologue-1 gene. Particularly the present disclosure provides systems, methods, and compositions for prime editing insertion or correction of mutations in the crumbs homologue-1 gene.

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: Compositions are described for direct protein delivery into multiple cell types in the mammalian inner ear. The compositions are used to deliver protein(s) (such as gene editing factors) editing of genetic mutations associated with deafness or associated disorders thereof. The delivery of genome editing proteins for gene editing and correction of genetic mutations protect or restore hearing from genetic deafness. Methods of treatment include the intracellular delivery of these molecules to a specific therapeutic target.

Abstract: Provided herein are systems, compositions, and methods for the incorporation of unnatural amino acids into proteins via nonsense suppression or rare codon suppression. Nonsense codons and rare codons may be introduced into the coding sequence of a protein of interest using a CRISPR/Cas9-based nucleobase editor described herein. The nucleobase editors are able to be programmed by guide nucleotide sequences to edit the target codons in the coding sequence of the protein of interest. Also provided are application enabled by the technology described herein.

Abstract: Aspects of the disclosure relate to Botulinum toxin X (BoNT X) protein variants (e.g., BoNT X protease variants). The variants provided herein have been evolved to cleave procaspase-1. Some of the variants provided herein do not cleave the native substrate of BoNT X protease, VAMP1 protein. Further aspects of the disclosure relate to nucleic acids encoding the procaspase-1 cleaving polypeptides described herein and expression vectors comprising the nucleic acids, as well as host cells and fusion proteins comprising the procaspase-1 cleaving polypeptides described herein and kits comprising the procaspase-1 cleaving polypeptides, nucleic acids, fusion proteins, expression vectors, or host cells described herein. Further aspects of the disclosure relate to methods of producing BoNT X protein variants (e.g., BoNT X protease variants) and methods of using the BoNT X protein variants (e.g., BoNT X protease variants), for example, to induce cell death.

Abstract: Aspects of this disclosure provide compositions, 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. Fusion proteins capable of inducing a cytosine (C) to guanine (G) change (i.e., transversion changes) in a nucleic acid (e.g., genomic DNA) are provided. Fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, base excision enzymes, and/or DNA repair proteins, are also provided. Methods for targeted nucleic acid editing are also provided. Reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are further provided in the present disclosure.

Abstract: Aspects of the disclosure relate to compositions, systems, and methods for evolving nucleic acids and proteins utilizing continuous directed evolution in the periplasm of a host cell. In some embodiments, the methods comprise passing a nucleic acid from cell-to-cell in a desired, function dependent manner. The linkage of the desired function and passage of the nucleic acid from cell-to-cell allows for continuous selection and mutation of the nucleic acid.

Abstract: Provided herein are systems, compositions, kits, and methods for the suppression of pain (e.g., chronic pain). Genes encoding ion channels (e.g., SCN9A) responsible for the propagation pain signals in neurons (e.g., DRG neurons) may be edited using a genome editing agent (e.g., a nucleobase editor). In some embodiments, loss-of-function ion channel mutants are generated, leading to pain suppression. In some embodiments, the genome editing agent is administered locally to the site of pain or to the nerves responsible for propagation of the pain signal.

Abstract: The disclosure provides amino acid sequence variants of Botulinum neurotoxin (BoNT) proteases that cleave (VAMP1, VAMP2, VAMP7, VAMP8, SNAP25, SNAP23, PTEN, etc.) and methods of evolving the same. In some embodiments, proteases described by the disclosure are useful for cleaving proteins found in a cell, that is in an intracellular environment. In some embodiments, proteases described by the disclosure are useful for treating diseases associated with increased or aberrant VAMP7, VAMP8, SNAP23 or PTEN expression or activity, for example, cancer and neurological disorders. Some aspects of this disclosure provide methods for generating BoNT protease variants by continuous directed evolution.

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 provided to induce cells of the inner ear to renter the cell cycle and to proliferate. In particular, hair cells are induced to proliferate by administration of a composition which activates the Myc and Notch. Supporting cells are induced to transdifferentiate to hair cells by inhibition of Myc and Notch activity or the activation of Atoh1. Methods of treatment include the intracellular delivery of these molecules to a specific therapeutic target.

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.