Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

A composition, engineering technology, applied in the direction of biochemical equipment and methods, other methods of inserting foreign genetic materials, drug combinations, etc.

Active Publication Date: 2015-12-16
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

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  • Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
  • Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
  • Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

Examples

Experimental program
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Effect test

example 1

[0751] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[0752] 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 non-coding RNAs, the pre-crRNA array and the 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 guides Cas9 to a DNA target consisting of the protospacer and the corresponding PA...

example 2

[0783] Example 2: CRISPR System Modifications and Alternatives

[0784] 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 metagenomes (see eg Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR loci can be grafted into t...

example 3

[0785] Example 3: Sample target sequence selection algorithm

[0786] 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 and CRISPR motif sequence (PAM) for the specified CRISPR enzyme. For example, the target site of Cas9 from S. pyogenes with the PAM sequence NGG can be identified by searching for 5'-Nx-NGG-3' on both the input sequence and the reverse complement of the input sequence. Likewise, a target site for Cas9 of S. pyogenes CRISPR1 with the PAM sequence NNAGAAW can be identified by searching for 5'-Nx-NNAGAAW-3' both on the input sequence and the reverse complement of the input sequence. Likewise, the target site of Cas9 of S. pyogenes CRISPR3 with the PAM sequence NGGNG can be identified by searching for 5'-Nx-NGGNG-3' on both the input sequence and the reverse complement of the input sequence. Can be fixed by program or specified by userN x The value "x...

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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 deliver 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 ceils 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 cited references [0002] This application claims the benefit of and priority to the following U.S. Provisional Patent Application Numbers: 61 / 736,527 filed December 12, 2012; 61 / 748,427 filed January 2, 2013; 61 / 758,468 filed January 30, 2013 , 61 / 769,046 filed February 25, 2013; 61 / 791,409 and 61 / 802,174 filed March 15, 2013, 61 / 806,375 filed March 28, 2013; 61 filed April 20, 2013 61 / 819,803 filed May 6, 2013; 61 / 828,130 filed May 28, 2013; 61 / 835,931 and 61 / 836,123 filed June 17, 2013 and July 17, 2013 61 / 847,537. [0003] Reference is also made to the following U.S. Provisional Patent Application Nos.: 61 / 799,800, filed March 15, 2013; 61 / 835,931, 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / 835,973; 61 / 862,468 and 61 / 862,355 filed August 5, 2013; 61 / 871,301 filed August 28, 2013; 61 / 960,777 filed September 25, 2013; and October 28, 2013 61 / 961,980 filed on date. [0004] The aforementioned applications, and all documents cited therein or durin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
CPCC12N15/102C12N15/63A01K67/0275C12N9/22C12N15/90A01K2217/052A01K2217/072A01K2227/105A01K2267/03C12N9/96A61P1/16A61P11/00A61P13/12A61P19/08A61P19/10A61P21/00A61P25/00A61P25/14A61P25/16A61P25/18A61P25/28A61P27/02A61P29/00A61P3/00A61P31/12A61P31/14A61P31/18A61P35/00A61P35/02A61P3/06A61P37/02A61P43/00A61P7/00A61P9/00C12N2800/22C12N15/85C12N15/907A01K2217/07C12N15/8509C12N15/86A01K2217/05C12N15/8213A01K67/0278C12N15/01A61K48/00
Inventor F.张M.海登雷希F.蓝L.斯维奇
Owner THE BROAD INST INC
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