Optimized non-canonical zinc finger proteins

A zinc finger protein, zinc finger technology, applied in chemical instruments and methods, peptides, depsipeptides, etc., can solve problems such as reduced ability

Active Publication Date: 2010-03-10
DOW AGROSCIENCES LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although zinc finger proteins containing these non-canonical fingers retain gene transcriptional regulatory functions, their ability to function as zinc finger nucleases (ZFNs) is in some cases protein decreased

Method used

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  • Optimized non-canonical zinc finger proteins
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  • Optimized non-canonical zinc finger proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0317] Embodiment 1: ZFN expression vector

[0318] A ZFN comprising encoding 4 fingers described in Examples 2 and 14 (referred to as "5 -8" and "5-9") sequence expression vector. Briefly, the 5-8 and 5-9 ZFNs (which comprise the nuclease domain of the type IIS restriction enzyme Fok I via a 4 amino acid ZC linker (Wah et al. (1998) Proc. Natl. Acad. Sci. USA 95 : 384-579 amino acids of the sequence of 10564-10569) fused four zinc finger domains) modified to CCHC structure. Additional modifications (substitutions and insertions) were also made to the residues between the C-terminal His and Cys zinc coordination structures and / or the C-terminal residues of the C-terminal Cys of Finger 2 and / or Finger 4.

Embodiment 2

[0319] Example 2: Gene Correction of eGFP in Reporter Cell Lines

[0320] ZFNs comprising CCHC zinc fingers described herein were tested for their ability to promote homologous recombination in the GFP system described in Urnov (2005) Nature 435(7042): 646-51 and U.S. Patent Publication No. 20050064474 (eg, Examples 6-11). ability. Briefly, 50 ng of each ZFN and 500 ng of the promoter-less GFP donor (Urnov (2005) Nature) were transfected into 500,000 reporter cells using 2 μL Lipofectamine 2000 per sample according to the Invitrogen Lipofectamine 2000 protocol.

[0321] Vinblastine at a final concentration of 0.2 μM was added 24 hours after transfection and removed 72 hours after transfection.

[0322] Cells were assayed for GFP expression 5 days after transfection by measuring 40,000 cells per transfection on a Guava benchtop FACS analyzer.

[0323] Such as figure 1 As shown, most ZFNs comprising the altered CCHC zinc fingers shown in Tables 1 and 2 above promote homologou...

Embodiment 3

[0324] Example 3: Editing of the chromosomal IL2Rγ gene by targeted recombination

[0325] The ZFNs described herein were also assayed in Urnov (2005) Nature 435(7042):646-51 and the endogenous IL2Rγ assay described in Example 2 of US Patent Publication No. 20050064474. Briefly, 2.5 μg of each ZFN expression construct was transfected into 500,000 K562 cells using Nucleofector (Amaxa). Genomic DNA was harvested and assayed for gene disruption at the endogenous IL2Rγ locus using the Surveyor Endonuclease Kit.

[0326] figure 2 ZFNs are shown on the upper left of . Specifically, altered zinc finger 20 refers to a CCHC zinc finger comprising the sequence HTRRCGLRGSQLV; zinc finger 21 comprises the sequence HAQRCGLRGSQLV (SEQ ID NO: 53); zinc finger 43 comprises the sequence HIRTCTGSQKP (SEQ ID NO: 75); zinc finger 45 comprises The sequence HIRTGCTGSQKP; zinc finger 47 comprises the sequence HIRRCTGSQKP; and zinc finger 48 comprises the sequence HIRRGCTGSQKP. Zinc fingers 20 a...

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Abstract

Disclosed herein are zinc fingers comprising CCHC zinc coordinating residues. Also described are zinc finger proteins and fusion proteins comprising these CCHC zinc fingers as well as polynucleotidesencoding these proteins. Methods of using these proteins for gene editing and gene regulation are also described.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application No. 60 / 874,911, filed December 14, 2006, and U.S. Provisional Application No. 60 / 932,497, filed May 30, 2007, the disclosures of which are hereby incorporated by reference in their entirety content. field of invention [0003] The present disclosure is in the fields of genome engineering, gene targeting, targeted chromosomal integration, protein expression, and epigenome editing. Background of the invention [0004] Sequence-specific binding of proteins to DNA, RNA, proteins and other molecules is involved in many cellular processes such as, for example, transcription, replication, chromatin structure, recombination, DNA repair, RNA processing and translation. The binding specificity of cellular binding proteins involved in protein-DNA, protein-RNA and protein-protein interactions contributes to development, differentiation and homeostasis. [0005] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/09C12N15/82C07K14/00
Inventor 其华·C·蔡杰弗里·米勒菲奥多·厄诺夫维普拉·K·舒克拉约瑟夫·F·皮托利诺莉萨·W·贝克罗比·J·加里森瑞安·C·布卢乔恩·C·米歇尔妮科尔·L·阿诺德萨拉·E·沃登
Owner DOW AGROSCIENCES LLC
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