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Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components

A technology of composition and use, applied in the direction of using vectors to introduce foreign genetic material, viruses, drug combinations, etc.

Active Publication Date: 2016-07-20
THE BROAD INST INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While genome editing technologies such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for generating targeted genome perturbations, there is still a need for affordable New genome engineering techniques that are easy to establish, scalable, and facilitate targeting multiple locations within the eukaryotic genome

Method used

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  • Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components
  • Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components
  • Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components

Examples

Experimental program
Comparison scheme
Effect test

example 1

[1057] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[1058] An exemplary type II CRISPR system is the type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, and two non-coding RNA elements tracrRNA and a short non-repetitive sequence. A characteristic array of repeats (direct repeats) spaced apart by segments (spacers, about 30 bp each). In this system, a targeted DNA double-strand break (DSB) is generated in four consecutive steps ( Figure 2A ). In the first step, two non-coding RNA pre-crRNA arrays and tracrRNA are transcribed from the CRISPR locus. In the second step, tracrRNA is hybridized to the direct repeat of pre-crRNA, and then processed into mature crRNA containing a separate spacer sequence. In the third step, the mature crRNA:tracrRNA complex guides Cas9 to the DNA target composed of the protospacer and the corresponding PAM by forming a heteroduplex between the spacer reg...

example 2

[1089] Example 2: CRISPR system modification and alternatives

[1090] The ability to use RNA programming sequence-specific DNA cleavage defines a new category of genome engineering tools for multiple research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. The optimized Cas9 activity can depend on the free Mg that is present in the nucleus of mammalian cells. 2+ High levels of free Mg 2+ (See, for example, Jinek et al., 2012, Science, 337:816), and preference for NGG motifs located just downstream of the protospacer restricts targeting of the human genome The average capacity per 12-bp in (Figure 9, evaluation of both the positive and negative strands of the human chromosomal sequence). Some of these constraints can be overcome by exploring the diversity of CRISPR loci across microbial metagenomics (see, for example, Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other ...

example 3

[1091] Example 3: Sample target sequence selection algorithm

[1092] A software program is designed to identify candidate CRISPR target sequences on both strands of the input DNA sequence based on the desired guide sequence length of the specified CRISPR enzyme and the CRISPR motif sequence (PAM). For example, you can search for 5’-N on both the input sequence and the reverse complement of the input sequence x -NGG-3' to identify the target site of NGG with PAM sequence of Cas9 from Streptococcus pyogenes. Similarly, you can search for 5’-N on both the input sequence and the reverse complement of the input sequence x -NNAGAAW-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR1 with PAM sequence NNAGAAW. Similarly, you can search for 5’-N on both the input sequence and the reverse complement of the input sequence x -NGGNG-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR3 with the PAM sequence NGGNG. Can be fixed by program or specified...

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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 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 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.

Description

[0001] Related applications and references [0002] The requirements are from the U.S. Provisional Patent Application 61 / 836,123 filed on June 17, 2013, 61 / 847,537 filed on July 17, 2013, 61 / 862,355 filed on August 5, 2013, and filed on August 28, 2013 61 / 871,301, 61 / 915,225 filed on December 12, 2013, 61 / 979,879 filed on April 15, 2014, and PCT / US2013 / 074667 filed on December 12, 2013 priority for U.S. purposes , This application is also a partial continuation application; and as permitted under US law, the US equivalent or the national phase to this point may further require and claim the priority of PCT / US2013 / 074667 and the requirements of PCT / US2013 / 074667 Priority of the application from the source of priority. [0003] The aforementioned applications, and all documents cited therein or during their examination procedures or ("application cited documents") and all documents cited or referenced in these applications cited documents, and all documents cited or referenced herein...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/10C40B40/08C12N9/22
CPCC12N9/22C12N15/1082C12N15/63C12Y301/00A61P43/00C12N15/86C12N2740/15041C12N2750/14141A01K67/0275A61K48/0058C12N15/907C12N2310/20C12N7/00C12N2740/15043C12N2750/14143
Inventor 张锋丛乐然霏M·海登里希L·斯维奇
Owner THE BROAD INST INC
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