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Genome editing using targeting endonucleases and single-stranded nucleic acids

一种单链核酸、靶向核酸的技术，应用在基因治疗、基因工程、植物基因改良等方向，能够解决低自发重组率、阻碍进展等问题

Inactive Publication Date: 2013-06-19
SIGMA ALDRICH CO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, low rates of spontaneous recombination in many cells and the scale of screening efforts and time required to isolate targeted events have hampered progress in this field

Method used

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  • Genome editing using targeting endonucleases and single-stranded nucleic acids
  • Genome editing using targeting endonucleases and single-stranded nucleic acids
  • Genome editing using targeting endonucleases and single-stranded nucleic acids

Examples

Experimental program
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Effect test

Embodiment 1

[0121] Example 1: Modification of RSK2 Kinase

[0122] The following examples detail the modification of the RSK2 kinase locus. Oligonucleotides (125nt) were designed to incorporate 3 different mutations into the RSK2 kinase chromosomal sequence. Oligonucleotides contain: (1) two point mutations in the ZFN binding site that prevent subsequent non-homologous end joining (NHEJ), (2) TGC to GTT change to switch Cys to Val, and (3) silencing Sex C to A change to generate a single BamHI site for clone screening ( figure 1 ). Oligonucleotides are generated using standard synthetic methods (eg, without chemical modification) and purified by PAGE. A pair of zinc finger nucleases (ZFNs) were designed to target the RSK2 kinase locus. A ZFN was designed to bind the sequence 5'-GTATACATAAAGCTA-3' (SEQ ID NO: 6; figure 1 left binding site shown in ), and another ZFN was designed to bind the sequence 5'-GGAGTTTGCAGTGAAGGTA-3' (SEQ ID NO: 7; figure 1 right binding site shown in ).

[...

Embodiment 2

[0125] Example 2: Modification of the AAVS1 locus

[0126] The following examples detail the use of oligonucleotides to introduce HindIII sites into the AAVS1 locus. Figure 4 The wild type sequence of the AAVS1 locus and the sequence (108 nt) of sense and antisense oligonucleotides containing HindIII sites are shown. These oligonucleotides were generated using standard methods (eg, without chemical modification) and purified by PAGE.

[0127] A pair of ZFNs were designed to target the AAVS1 locus. A ZFN was designed to bind the sequence 5'-ACCCACACAGTGG-3' (SEQ ID NO: 8; Figure 4 left binding site shown in ), and another ZFN was designed to bind the sequence 5'-TAGGGACAGGAT-3' (SEQ ID NO: 9; Figure 4 right binding site shown in ). Capped, polyadenylated mRNAs encoding ZFNs were prepared using standard methods. ZFN mRNA and HindIII site containing oligonucleotides were nucleoffected into K562, HCT116, U2OS, A549, HEK293, HepG2 or MSF7 cells. After a period of incubatio...

Embodiment 3

[0129] Example 3: Modification of the AAVS1 locus - length of oligonucleotides

[0130] To determine whether shorter oligonucleotides might be used to deliver HindIII sites to the AAVS1 locus, oligonucleotides ranging in length from 36 to 106 nt were prepared. For example, a 106 nt oligonucleotide has 50 nt sequence identity (ie, has 50 nt homology arms) on either side of the ZFN cleavage site, with a HindIII site (6 nt) between the homology arms. The sequences of the oligonucleotides are shown in Table 1 below.

[0131]

[0132] K562 cells were nucleoffected with 2.5 μg of plasmid DNA encoding each ZFN directed to AAVS1 (5 μg total) and 3 μl of a 100 μM oligonucleotide stock (sense or antisense). Cells were harvested 2 days after nucleofection. Genomic DNA was PCR amplified and digested with HindIII. Figure 7 Integration of sense strand AAVS1-HindIII oligonucleotides of different lengths is demonstrated. Delivery of the ZFN and either oligonucleotide results in integr...

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Abstract

The present invention provides methods and kits for editing specific chromosomal sequences in cells. In particular, targeting endonucleases and single-stranded nucleic acids are used to edit the chromosomal sequence.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 61 / 410,124, filed November 4, 2010, U.S. Provisional Application No. 61 / 382,965, filed September 15, 2010, and U.S. Provisional Application No. 61 / 367,022, filed July 23, 2010 , each of which is hereby incorporated by reference in its entirety. field of invention [0003] The present invention generally relates to the editing of specific chromosomal sequences using targeting endonucleases and single-stranded nucleic acids. Background of the invention [0004] Routine genome engineering has great potential throughout basic research, drug discovery, and cell-based medicines. Many existing methods for targeted gene knockout, mutagenesis or integration rely on homologous recombination. However, low rates of spontaneous recombination in many cells and the scale of screening efforts and time required to isolate targeted events have hampered progress in this field. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00A61K48/00
CPCC12N9/22C12N15/907A61K48/00C12N15/00C12N15/09C12Q1/6869C12Q2521/301C12N15/85
Inventor 陈福强S.M.普吕特-米勒G.D.戴维斯
Owner SIGMA ALDRICH CO LLC
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