Abstract: Provided are compositions and methods for precise genome editing. The compositions include a fusion protein comprising a T4 DNA polymerase segment and a segment of an MS2 bacteriophage coat protein. The fusion protein operates with a Cas enzyme and one or more guide RNAs to produce one or more indels. The indel is produced in a DNA repair template free manner. Methods for producing the indels are also provided. A method includes introducing into the cell a fusion protein containing a T4 DNA polymerase segment and a segment of an MS2 bacteriophage coat protein, a Cas enzyme, and a guide RNA comprising MS2 protein binding sites. The guide RNA directs the Cas enzyme, the T4 DNA polymerase and the MS2 binding protein to the selected chromosome locus to produce the indel. The indel may correct a mutation in an open reading frame encoded by the selected chromosome locus.

Abstract: Provided are compositions and methods that include an engineered DNA polymerase used in combination with a Cas9 protein. The combination exhibits improved on-target chromosomal alterations, increases the proportion of precise 1- to 3-base-pair insertions at target sites, and reduces translocations caused by previously available systems.

Abstract: Provided are compositions and methods used for precise template-free correction of BRCA1 mutation in human cells via genome editing. The method involves modifying DNA that includes a BRCA1-5382-InsC mutation by introducing into cells comprising the BRCA1-5382-InsC a Cas enzyme and a guide RNA. A guide RNA that produces improved results relative to other guide RNAs is provided. Also provided are modified stem cells that contain an introduced BRCA1-5382-InsC mutation.

Abstract: Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. The disclosure reports CRISPR/Cas9-mediated gene editing (Myo-editing) is effective at correcting the dystrophin gene mutation in the mdx mice, a model for DMD. Further, the disclosure reports optimization of germline editing of mdx mice by engineering the permanent skipping of mutant exon (exon 23) and extending exon skipping to also correct the disease by post-natal delivery of adeno-associate virus (AAV). AAV-mediated Myo-editing can efficiently rescue the reading frame of dystrophin in mdx mice in vivo. The disclosure reports means of Myo-editing-mediated exon skipping has been successfully advanced from somatic tissues in mice to human DMD patients-derived iPSCs (induced pluripotent stem cells).

Abstract: The disclosure provides a method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a Cas9 nuclease or a sequence encoding a Cas9 nuclease, and a gRNA or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene. The administering restores dystrophin expression in at least a subset of the subjects cardiomyocytes, and may at least partially or fully restore cardiac contractility.

Abstract: Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. The disclosure reports CRISPR/Cpf1-mediated gene editing (Myo-editing) is effective at correcting the dystrophin gene mutation in the mdx mice, a model for DMD. Further, the disclosure reports optimization of germline editing of mdx mice by engineering the permanent skipping of mutant exon and extending exon skipping to also correct the disease by post-natal delivery of adeno-associated virus (AAV). AAV-mediated Myo-editing can efficiently rescue the reading frame of dystrophin in mdx mice in vivo. The disclosure reports means of Myo-editing-mediated exon skipping has been successfully advanced from somatic tissues in mice to human DMD patients-derived iPSCs (induced pluripotent stem cells).

Abstract: CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. In vivo AAV-mediated delivery of gene-editing components machinery has been shown to successfully remove mutant sequence to generate an exon skipping in the cardiac and skeletal muscle cells of postnatal mdx mice, a model of DMD. Using different modes of AAV9 delivery, the restoration of dystrophin protein expression in cardiac and skeletal muscle of mdx mice was achieved. Here, a humanized mouse model for DMD is created to help test the efficacy of genome editing to cure DMD. Additionally, to facilitate the analysis of exon skipping strategies in vivo in a non-invasive way, a reporter luciferase knock-in version of the mouse model was prepared. These humanized mouse models provide the ability to study correcting of mutations responsible for DMD in vivo.

Abstract: Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. The disclosure reports CRISPR/Cas9-mediated gene editing (Myo-editing) is effective at correcting the dystrophin gene mutation in the mdx mice, a model for DMD. Further, the disclosure reports optimization of germline editing of mdx mice by engineering the permanent skipping of mutant exon (exon 23) and extending exon skipping to also correct the disease by post-natal delivery of adeno-associate virus (AAV). AAV-mediated Myo-editing can efficiently rescue the reading frame of dystrophin in mdx mice in vivo. The disclosure reports means of Myo-editing-mediated exon skipping has been successfully advanced from somatic tissues in mice to human DMD patients-derived iPSCs (induced pluripotent stem cells).