Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells

A technology of mitosis and composition, applied in the manufacture of gene therapy compositions, biochemical equipment and methods, drug combinations, etc.

Inactive Publication Date: 2016-06-15
THE BROAD INST INC +1
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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, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells
  • Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells
  • Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

[1012] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[1013] 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 guides Cas9 to a DNA target consisting of the protospacer and the corresponding PAM via het...

example 2

[1044] Example 2: CRISPR System Modifications and Alternatives

[1045] 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

[1046] Example 3: Sample target sequence selection algorithm

[1047] 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, 5'-N can be obtained by searching both the input sequence and the reverse x -NGG-3' to identify the target site of Cas9 from Streptococcus pyogenes with the PAM sequence NGG. Likewise, 5'-N can be searched both on the input sequence and the reverse x -NNAGAAW-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR1 with the PAM sequence NNAGAAW. Likewise, 5'-N can be searched both on the input sequence and the reverse 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 by user N x The value "x" in , such as 20.

[1048] Since multiple occurrences in the gen...

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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 include post mitotic cells 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 cited references [0002] Claims from U.S. Provisional Patent Applications 61 / 836,123 filed June 17, 2013, 61 / 847,537 filed July 17, 2013, 61 / 862,355 filed August 5, 2013, August 28, 2013 61 / 871,301, 61 / 915,203 filed December 12, 2013, 61 / 979,573 filed April 15, 2014, and PCT / US2013 / 074667 filed December 12, 2013 Priority for U.S. purposes right, this application is also a continuation-in-part; and as may be permitted under US law, US equivalents or national phases hereto may further claim and claim priority to PCT / US2013 / 074667 and to PCT / US2013 / 074667 Priority of an application from the source from which priority is claimed. [0003] The aforementioned applications, and all documents cited therein or during their prosecution or ("Application Citations") and all documents cited or referenced in these Application Citations, and all documents cited or referenced herein ( "documents cited herein"), all documents cited or referenced in documents ci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/10C12N9/22
CPCC12N9/22C12N15/1082C12N15/63C12N15/102C12Y301/00A61P1/00A61P11/00A61P13/00A61P17/00A61P19/00A61P21/00A61P25/04A61P25/16A61P25/28A61P31/00A61P9/00A01K67/0275A61K48/0058A61K48/0091C12N15/86C12N15/907C12N7/00A01K2217/052A61K48/00C12N2750/14132C12N2750/14152
Inventor 张锋M·海登里希L·斯维奇
Owner THE BROAD INST INC
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