CRISPR-Cas systems and methods for altering expression of gene products

A gene product, genome technology, applied in the field of CRISPR-CAS systems and methods for altering the expression of gene products

Active Publication Date: 2017-03-08
THE BROAD INST INC +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • CRISPR-Cas systems and methods for altering expression of gene products
  • CRISPR-Cas systems and methods for altering expression of gene products
  • CRISPR-Cas systems and methods for altering expression of gene products

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[0168] The following examples are given for the purpose of illustrating different embodiments of the invention and are not intended to limit the invention in any way. The inventive examples, along with the methods described herein, presently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Variations therein and other uses encompassed within the spirit of the invention as defined by the scope of the claims will occur to those of ordinary skill in the art.

example 1

[0169] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[0170] An exemplary type II CRISPR system is the type II CRISPR locus from S. pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1 and two non-coding RNA elements tracrRNA and a short sequence composed of non-repetitive sequences. A characteristic array of repeated sequences (direct repeats) spaced apart by segments (spacers, about 30 bp each). In this system, targeted DNA double-strand breaks (DSBs) are generated in four sequential steps ( Figure 2A ). In the first step, two noncoding RNAs, pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. In the second step, the tracrRNA is hybridized to the direct repeat of the pre-crRNA, which is then processed into a mature crRNA containing a separate spacer sequence. In the third step, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via ...

example 2

[0200] Example 2: CRISPR System Modifications and Alternatives

[0201] The ability to program sequence-specific DNA cleavage using RNA defines a new class of genome engineering tools for a variety of research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity can be dependent on free Mg present in mammalian nuclei 2+ High levels of free Mg 2+ Availability of NGG motifs (see e.g. Jinek et al., 2012, Science, 337:816), and a preference for NGG motifs located just downstream of protospacers limits targeting to the human genome average capacity per 12-bp in (Fig. 9, both positive and negative strands of human chromosomal sequences were evaluated). Some of these constraints can be overcome by exploring the diversity of CRISPR loci across microbial metagenomics (see e.g. Makarova et al., 2011, Nat Rev Microbiol, 9:467) . Other CRISPR loci can be grafted ...

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Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.

Description

[0001] This application is a divisional application of the national application number 201380072715.1 (PCT / US2013 / 074743) filed on December 12, 2013 entitled "CRISPR-CAS system and method for altering the expression of gene products" . [0002] Related applications and cited references [0003] This application claims the title "CRISPR-Cas System and Method for Altering the Expression of a Gene Product (CRISPR-Cas. CAS SYSTEMSAND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS)" US provisional patent application 61 / 842,322 and US patent application 14 / 054,414 priority. Filed on December 12, 2012, January 2, 2013, March 15, 2013, and June 17, 2013, respectively, with Bode Reference No. BI-2011 / 008 / WSGR Docket No. 44063- 701.101, BI-2011 / 008 / WSGR Docket No. 44063-701.102, Broad Reference No. BI-2011 / 008 / VP Docket No. 44790.02.2003, and BI-2011 / 008 / VP Docket No. 44790.03.2003, all titled "Use Priority claims to U.S. Provisional Patent Applications 61 / 736,527, 61 / 748,427, 61 / 791...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N9/22C12N9/96C12N15/10C12N15/11
CPCC12N9/22C12N9/96C12N15/102C12N15/111C12N15/63C12N2310/10C12N15/1082A61K38/43A61K38/46A61K38/47C12N2310/20C12N15/902C12N15/85C12N15/907C12N9/14C12N9/52C12Q1/68C12N15/00C12N2800/80C12N2800/30C12N2800/90
Inventor F.张
Owner THE BROAD INST INC
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