Abstract: The present invention is related to the field of genetic engineering. In particular, the repair, reversion and/or conversion of genetic mutations that are linked to a muscular dystrophy disease. Specifically contemplated are gene editor nuclease proteins or base editor proteins that are targeted to the muscular dystrophy genetic mutations or pathogenic variants. Such gene editor nuclease proteins include, but are not limited to Cas12a nuclease proteins and adenine base editor proteins. Repair, reversion and/or disruption of the genetic mutation or pathogenic variant reduces at least one symptom of a muscular dystrophy disease.

Abstract: The present applications discloses an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency exemplified by endogenous loci in cultured cell lines. Using this genome modification system, tumor formation can be initiated through somatic cell editing in the adult mouse. Furthermore, a dual adeno-associated vims (AAVs) is utilized for the delivery of a split-intein prime editor for correction of in vivo pathogenic mutations.

Abstract: In some aspects, the disclosure relates to recombinant adeno-associated viruses (rAAVs) comprising a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transcriptional regulator domain and methods of using the same. In some embodiments, expression of the fusion protein results in modified expression of a target gene in a cell.

Abstract: RNA-guided programmable cytosine and adenine base editors are a powerful class of genome editing tool for the introduction of localized base transitions without generating a double-stranded DNA break. Base editors (BE) have an optimal window of activity relative to the PAM recognized by the Cas9 enzyme and these constructs are strand selective. Here we demonstrate that fusion of a programmable DNA-binding domain (pDBD) or another Cas9 orthologue to spCas9-BE, we can produce an RNA-programmable Cas9-BE-pDBD chimera or Cas9-BE-Cas9 chimeras with dramatically improved activities and increased targeting range. Cas9-pDBD or Cas9-Cas9 fusion base editors display an expanded targeting repertoire and achieve highly specific genome editing, which can be tailored to achieve extremely precise genome editing at nearly any genomic locus.

Abstract: The present invention is related to the field of gene editing. In particular, the gene editing is directed toward single nucleotide base editing. For example, such single nucleotide base editing results in a conversion of a OG base pair to a T*A base pair. The high accuracy and precision of the presently disclosed single nucleotide base gene editor is accomplished by an NmeCas9 nuclease that is fused to a nucleotide deaminase protein. The compact nature of the NmeCas9 coupled with a larger number of compatible protospacer adjacent motifs provide the Cas9 fusion constructs contemplated herein to have a gene editing window that can edit sites that are not targetable by other conventional SpyCas9 base editor platforms.

Abstract: In some aspects, the disclosure relates to recombinant adeno-associated viruses (rAAVs) comprising a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transcriptional regulator domain and methods of using the same. In some embodiments, expression of the fusion protein results in modified expression of a target gene in a cell.

Abstract: Provided herein are ribonucleoprotein (RNP) complexes comprising a DNA-targeting endonuclease Cas (CRISPR-associated) protein and a guide RNA (gRNA) that that targets and hybridizes to the ?-Globin gene. In one embodiment, the Cas protein is Cas9 and the gRNA comprises the sequence of SEQ ID NO: 1. In one embodiment, the Cas protein is Cas12a and the gRNA comprises the sequence of SEQ ID NO: 3.

Abstract: Provided herein are reagents and methods for targeting the ELANE gene for inhibition. Further provided herein is a method for producing a progenitor cell or a population of progenitor cells having decreased ELANE mRNA or protein expression.

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage.

Abstract: Chemically modified crRNAs and tracrRNAs are provided. crRNAs and tracrRNAs with 5? and/or 3? conjugated moieties are provided. crRNAs and tracrRNAs with modifications in the repeat region of the crRNA or the anti-repeat region of the tracrRNA are provided. Methods of using the crRNAs and tracrRNAs for genome editing with a CRISPR nuclease and kits for performing the same are also provided.

Abstract: The present invention is directed to the filed of gene therapy. In particular, compositions and methods are disclosed that repair gene microduplication mutations by reversion to a wild type sequence. For example, the creation of a double stranded break by a programmable nuclease protein within a microduplication induces the microhomology mediated end joining DNA repair pathway that in the process of DNA repair removes the microduplication mutation and restores the wild type sequence.

Abstract: Provided herein are synthetic nucleic acids that encode a CRISPR enzyme fused to a series of nuclear localization signal sequences for the use of altering expression of a gene in a cell. Further provided herein are methods for altering gene expression in a cell comprising introducing the synthetic nucleic acids and a guide RNA, or a polypeptide and a guide RNA into a cell. In certain embodiments the cell is a quiescent cell, for example, a hematopoietic stem cell.

Abstract: Provided herein are methods and compositions for increasing fetal hemoglobin levels in a cell by disrupting BCL11A expression at the genomic level. Also provided herein are methods and compositions relating to the treatment of hemoglobinopathies by reinduction of fetal hemoglobin levels.

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage.

Abstract: The invention is based on the discovery that one can create a drug-regulated gene regulation system by creating a fusion polypeptide or chimera that includes three portions: a zinc finger (ZF) protein that recognizes and binds to a nucleic acid recognition sequence in the vicinity of a promoter of the target gene, (ii) a dimerization domain of a tetracycline repressor, which contains a drug-dependent switch, and (iii) an accessory domain to alter the expression of the target gene (such as an activation or repression domain). This chimera is administered to, or expressed in, a cell harboring the target gene to be regulated.

Abstract: The present application relates to the hybrid selection methods in prokaryotes using counterselectable reporter genes.

Abstract: The invention is based on the discovery that one can create a drug-regulated gene regulation system by creating a fusion polypeptide or chimera that includes three portions: a zinc finger (ZF) protein that recognizes and binds to a nucleic acid recognition sequence in the vicinity of a promoter of the target gene, (ii) a dimerization domain of a tetrac repressor, which contains a drug-dependent switch, and (iii) an accessory domain to alter the expression of the target gene (such as an activation or repression domain). This chimera is administered to, or expressed in, a cell harboring the target gene to be regulated.