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TAL effector-mediated DNA modification

一种效应子、特异性的技术，应用在使用转录激活因子样效应子序列领域，能够解决有效方法难以实现等问题

Active Publication Date: 2017-06-13
RGT UNIV OF MINNESOTA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

An effective approach to this gene targeting is elusive

Method used

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  • TAL effector-mediated DNA modification
  • TAL effector-mediated DNA modification
  • TAL effector-mediated DNA modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0211] Example 1 - Determining the code for TAL effector-DNA recognition

[0212] To determine whether there is a one-to-one linear correspondence between consecutive nucleotides in the RVD and TAL target sites, the TAL effector RVD sequences were used to scan the predicted promoter regions of each of the 10 TAL effector known target genes (i.e. 1,000 bp in front of the translation start site is shown) for alignments that minimize entropy in RVD nucleotide associations (arbitrary). The following formula is used to quantify entropy, where R is the RVD group of effectors, D is the group of 4 nucleotides (A, C, G, T), and fi,j represents the observed i-th RVD associated with j-th Nucleotide frequency:

[0213]

[0214] Various low-entropy sites exist in each promoter. For the effector AvrBs3, however, only one mapped to the 54bp upa20 promoter fragment previously identified as necessary and sufficient for activation and which is consistent with the UPA box common to genes ...

Embodiment 2

[0239] Example 2 - TALEN can work in yeast

[0240] Plasmid construction: The protein coding sequence of TAL effector AvrBs3 was obtained by digesting the plasmid with BamHI. The DNA segment primarily encoding the repeat domain was cleaved with SphI. The amino acid sequence of AvrBs3 can be found in GENBANK accession number P14727 and SEQ ID NO: 12 ( image 3 ), the nucleotide sequence is found in Accession No. X16130 and SEQ ID NO: 13 ( Figure 4 )middle. exist Figure 4 , BamHI and SphI sites are shown in bold and underlined. The AvrBs3 BamHI and SphI fragments were cloned into the nuclease expression vector pDW1789_TAL (Fig. 5), adjacent to the sequence encoding the FokI nuclease domain. To clone the AvrBs3 target site into the target reporter plasmid, two complementary DNA oligomers containing two AvrBs3 recognition sites arranged in reverse and an 18 bp spacer sequence between them were synthesized at the 5' and 3' ends, respectively. Has BglII and SpeI prominentl...

Embodiment 3

[0245] Example 3 - Modular Assembly of TAL Effector Repeats for Custom TALENs

[0246] Complementary oligonucleotides of 102 base pairs corresponding to each of the four individual TAL effector repeats, each assigned a different nucleotide, were synthesized, annealed, and either individually or in 2 and 3 of all permutations Combinatorial cloning of repeat sequences into high-copy bacterial cloning vectors was performed using standard restriction digestion and ligation techniques to generate 4 single repeat modules, 16 double repeat modules, and 64 triple repeat modules (e.g. Figure 11 shown). The desired TAL effector coding sequence was assembled by introducing the appropriate modular sequence into a Gateway-ready high-copy bacterial cloning vector comprising a truncated version of the tal1c gene that lacks the central repeat region except for the final characteristic half repeat. For example, an 18-repeat TAL effector coding sequence can be assembled by sequentially int...

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Abstract

Materials and methods related to gene targeting (e.g., gene targeting with transcription activator-like effector nucleases; ''TALENS'') are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application Serial No. 61 / 285,324 filed December 10, 2009, U.S. Provisional Application Serial No. 61 / 352,108 filed June 7, 2010, and U.S. Provisional Application Serial No. 61 / 352,108 filed July 22, 2010 Priority of 61 / 366,685, all applications are hereby incorporated by reference in their entirety. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with government support under Grant Nos. 0820831 and 0504304 awarded by the National Science Foundation. The government has certain rights in this invention. technical field [0005] The present invention relates to methods of gene targeting, particularly methods involving the use of transcription activator-like (TAL) effector sequences. Background technique [0006] The ability to modify chromosomes through homologous recombination (gene targeting) has long been a struggle for biologists. Fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N9/16A61K48/00A61P31/12
CPCA61K38/465C12N9/16C12N15/63A61K48/00C12N15/102C12N15/1082C12Y301/21004A61P31/12C12N15/62C12N9/22C12N15/01C12N15/09C12N15/10C12N15/11C12N15/64C12N15/8213C12N9/14C12N15/907C12N2810/85C12N9/0022
Inventor D·F·沃塔斯A·波格丹诺维张峰M·克里斯蒂安T·瑟马克C·L·施米特E·多伊尔王俐
Owner RGT UNIV OF MINNESOTA
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