Thermostable Cas9 nucleases. The present invention relates to the field of genetic engineering and more particularly to nucleic acid editing and genome modification. The present invention provides an isolated Cas protein or polypeptide fragment thereof having an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% identity therewith. The Cas protein or polypeptide is capable of binding, cleaving, marking or modifying a double stranded target polynucleotide at a temperature in the range 20° C. and 100° C. inclusive. The invention further provides isolated nucleic acid molecules encoding the Cas9 nucleases, expression vectors and host cells. The invention also provides PAM sequences recognized by the Cas protein or polypeptide, The Cas9 nucleases disclosed herein provide novel tools for genetic engineering in general, in particular at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.

The disclosure provides methods for carrying out Real Time Cellular Thermal Shift Assays (RT-CETSA). Also provided are molecular constructs and protein constructs for use in such assays and devices suitable for carrying out such assays.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

We disclose dumbbell-shaped vectors adapted for efficient expression in mammalian cells. We also disclose a novel method allowing the efficient synthesis of dumbbell-shaped vectors at low cost for delivery of recombinant DNA and RNA into host cells; and the use of dumbbell-shaped vectors for transient expression in, for example, primary human cells.

The invention provides for recombinant AAV vectors comprising a miniaturized human micro-dystrophin gene and methods of using the recombinant vectors to reduce or prevent fibrosis in subjects suffering from muscular dystrophy.

In one embodiment, the invention provides a dual-cassette gene vehicle comprising cassettes for expression of both a mini-dystrophin gene and NF-κB/p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue, which is an adeno-associated viral (AAV) vector, wherein the mini-dystrophin gene is operably linked to a construct comprising a muscle-specific first promoter and a modified Mcken (MCK) enhancer and wherein the NF-κB/p65-shRNA gene is under the control of a second promoter. Also are provided pharmaceutical compositions comprising such gene vehicles and a method for ameliorating Duchenne muscular dystrophy (DMD) employing such gene delivery vehicles and pharmaceutical compositions.

We describe a Flt-1 decoy comprising a VEGF binding domain of Flt-1 and a membrane anchoring domain, in which the Flt-1 decoy does not substantially comprise an intracellular domain.

Compositions and methods for treating a mammalian subject for an optic nerve (ON) neuropathy and/or reducing or ameliorating degeneration of axons and/or soma of RGCs are provided. Aspects of the composition include a mammalian viral vector, comprising a murine g-synuclein promoter, or functional fragment thereof, that promotes expression of a transgene specifically in retinal ganglion cells (RGCs), said promoter in operable linkage with an expression cassette encoding the transgene, wherein the expressed transgene inhibits activity of an expression product of an endogenous pro-neurodegenerative gene involved in an ER stress and/or UPR pathway that leads to axon or soma degeneration in the RGCs. Aspects of the methods include intravitreally administering the composition to treat the subject for the ON neuropathy. A variety of ON neuropathies may be treated by practicing the methods, including retinal ganglion cell degeneration, glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases.

The present invention relates to the delivery of oligomers for treating patients with a 5′ mutation in their DMD gene other than a DMD exon 2 duplication. The invention provides methods and materials for activating an internal ribosome entry site in exon 5 of the DMD gene resulting in translation of a functional truncated isoform of dystrophin. The methods and materials can be used for the treatment of muscular dystrophies arising from 5′ mutations in the DMD gene such as Duchenne Muscular Dystrophy or Becker Muscular Dystrophy.

Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.