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

A composite, engineered technology, applied in other methods of inserting foreign genetic material, digestive system, respiratory diseases, etc.

Pending Publication Date: 2020-03-10
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, transcriptional activator-like effectors (TALEs), or homing meganucleases are available for generating targeted genome perturbations, affordable New genome engineering technology that is easy to establish, scalable, and convenient for targeting multiple locations within the eukaryotic genome

Method used

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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

[0863] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[0864] 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 non-repetitive sequence. 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 PAM via formation of a he...

example 2

[0895] Example 2: CRISPR System Modifications and Alternatives

[0896] 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+(See e.g. Jinek et al., 2012, Science, 337:816), and the preference for NGG motifs located just downstream of the protospacer 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 into the mammalian cell...

example 3

[0897] Example 3: Sample target sequence selection algorithm

[0898] 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, the target site of Cas9 of S. pyogenes CRISPR1 with the PAM sequence NNAGAAW can be identified by searching for 5'-Nx-NNAGAAW-3' on both 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 user N x The value ...

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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] This application is a subsection of an invention patent application with an application date of December 12, 2013, an application number of 2013800727522, and an invention title of "Delivery, Engineering and Optimization of Systems, Methods and Compositions for Sequence Manipulation and Therapeutic Applications" case application. [0002] Related applications and cited references [0003] This application claims benefit 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 / 814,263; 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. [0004] Reference is also made to the following U.S. Provisional Patent Application Nos.:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/113C12N15/864C12N15/867C12N5/10C12N15/90A61K31/7088A61P35/00A61P35/02A61P29/00A61P37/02A61P3/00A61P1/16A61P25/18A61P11/00A61P13/12A61P21/00A61P19/08A61P25/00A61P3/06A61P7/00A61P31/12A61P31/14A61P31/18A61P9/00A61P19/10A61P25/28A61P25/14A61P25/16A61P27/02
CPCC12N15/102C12N15/63A01K67/0275C12N9/22C12N15/90A01K2217/052A01K2217/072A01K2227/105A01K2267/03C12N9/96A61P1/16A61P11/00A61P13/12A61P19/08A61P19/10A61P21/00A61P25/00A61P25/14A61P25/16A61P25/18A61P25/28A61P27/02A61P29/00A61P3/00A61P31/12A61P31/14A61P31/18A61P35/00A61P35/02A61P3/06A61P37/02A61P43/00A61P7/00A61P9/00C12N15/113C12N15/907C12N2310/10C12N2310/20C12N2320/11C12N2320/30C12N2750/14143G16B20/00G16B20/20G16B20/30G16B20/50G16B30/00G16B30/10C12N2800/22C12N15/85A01K2217/07C12N15/8509C12N15/86A01K2217/05C12N15/8213A01K67/0278C12N15/01A61K48/00
Inventor F.张M.海登雷希F.蓝L.斯维奇
Owner THE BROAD INST INC
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