US 2016/0354487 A1Published application

Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders

AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology, University of Iowa Research Foundation

InventorsFeng Zhang, Beverly Davidson, Chie-yu Lin, Edgardo Rodriguez

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Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a SIN CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing SIN CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

A non-naturally occurring or engineered self-inactivating CRISPR-Cas composition comprising:

I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) at least one first guide sequence capable of hybridizing to a target DNA,
(b) at least one tracr mate sequence, and
(c) at least one tracr sequence, and
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,
II. a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme,
wherein parts I and II comprise a first CRISPR-Cas system, and wherein,
III. the composition further comprises
(a) at least one second guide sequence capable of hybridizing to a sequence in or of the CRISPR-Cas system,
(b) at least one tracr mate sequence, and
(c) at least one tracr sequence, and
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, wherein parts I and III comprise a second CRISPR-Cas system,
and said composition when transcribed comprises
a first CRISPR complex comprising the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or is hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
a second CRISPR complex comprising a CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to a sequence of a polynucleotide comprising or encoding the CRISPR-Cas system, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
wherein the first guide sequence directs sequence-specific binding of a first CRISPR complex to the target DNA, and
wherein the second guide sequence directs sequence-specific binding of a second CRISPR complex to a sequence comprising a polynucleotide comprising or encoding a component of the CRISPR-Cas system and whereby there is diminished activity of the first CRISPR-Cas system over a period of time, and the CRISPR-Cas composition is self-inactivating (“SIN CRISPR-Cas composition”).

14 more elements

Facts

Application

15/179,941Filed 10 Jun 2016

Priority

12 Dec 2013

Published

8 Dec 2016

Term ends

Not estimatedThe source records don’t support an estimate.

Classification33 codes

C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
C12N 15/63
Recombinant DNA-technology: introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
A01K 67/0276
Genetically modified vertebrates, e.g. transgenic: knock-out vertebrates
A61K 31/713
Compounds having three or more nucleosides or nucleotides: double-stranded nucleic acids or oligonucleotides

Family

Application61/915,150Us provisional application · filed 12 Dec 2013

Application62/010,879Us provisional application · filed 11 Jun 2014

Application62/010,888Us provisional application · filed 11 Jun 2014

Application15/179,941Continuation in part

US 2016/0354487 A1This patent

From the US filing history. Foreign family members aren’t listed.

Abstract

Claims40