Nucleic acids, compositions and methods for the excision of target nucleic acids

A technology for target nucleic acid and endonuclease nucleic acid, which is applied in the fields of nucleic acids, compositions and methods for excising target nucleic acid, and can solve the problems of low frequency of excision events and the like

Active Publication Date: 2013-07-31
AMYRIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other methods produce excision events at a lower frequency, so it is necessary

Method used

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  • Nucleic acids, compositions and methods for the excision of target nucleic acids
  • Nucleic acids, compositions and methods for the excision of target nucleic acids
  • Nucleic acids, compositions and methods for the excision of target nucleic acids

Examples

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Embodiment 1

[0125] 6.1 Example 1: Construction of x-marked construct

[0126] The compositions and methods described in this application are used to prepare and identify a series of resectable selectable markers for Saccharomyces cerevisiae, which are referred to as "x-markers" in this application. The first generation of markers uses I-SceI endonuclease, and the parameters of the DNA constructs tested are shown in figure 1 The parameters include: (1) the different lengths of the flanks of the forward repeat of the endonuclease site and (2) the comparison of one and two endonuclease sites. The second-generation marker uses the results of the first-generation test, which broadens the application of I-SceI endonuclease. The third-generation x-tag has been proven to be effective and can be extended to other endonucleases listed in Table 1 below.

[0127] Table 1: Recognition and cleavage sites of endonucleases.

[0128]

[0129] The reagents used in the experiment are described below, and the re...

Embodiment 2

[0191] 6.2 Example 2: Excision of chromosomal DNA selectable marker

[0192] This example demonstrates that the x-marker construct described in Example 1 can mediate the excision of the selectable marker from the chromosomal DNA of the host cell. As described below, transfer the constructs into cells, inoculate the cells in a medium selective for x-marking, and confirm the correct integration by colony PCR; the treatment of such strains is the same as any other preparations prepared with standard markers The strain is the same, the x mark can exist stably. Third, when the x-mark needs to be removed, a single copy (CEN.ARS) plasmid containing its own marker and a homing endonuclease gene expression construct is used to transform the strain, as described in the following section. After selecting the existing plasmids for several generations and inducing the expression of the homing endonuclease gene, the loss of the x mark in the strain is detected. Finally, the strain is grown...

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Abstract

Nucleic acids, compositions, and methods that allow for the excision of one or more loci from the genome of a host cell are provided herein. In particular, provided herein is an excisable nucleic acid construct comprising, in a 5' to 3' orientation: a first tandem repeat nucleic acid, a first homing endonuclease recognition site, a target nucleic acid, a second homing endonuclease recognition site, and a second tandem repeat nucleic acid. In some embodiments, the excisable nucleic acid construct is integrated into the host cell genome, and the target nucleic acid can be excised from the host cell genome by contacting the homing endonuclease recognition sites with one or more appropriate homing endonucleases.

Description

[0001] This application claims the priority of U.S. Provisional Application No. 61 / 378,350 whose filing date is August 30, 2010, the entire content of which is incorporated into this application by reference. 1. Technical Field [0002] The nucleic acids, compositions, and methods provided in this application generally involve the fields of molecular biology and genetic engineering. 2. Background technology [0003] In many fields, including metabolic engineering, industrial microbiology, synthetic biology, and basic molecular genetics research, it is necessary to use genetic engineering techniques to excise target nucleic acids from the host cell genome or episomal genes. However, the methods previously used to remove target nucleic acids have been restricted and limited in application. For example, the use of site-specific recombinase methods to remove harmful specific recombinase binding sites will leave behind, and these sites will create potential genomic instability in the h...

Claims

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Application Information

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IPC IPC(8): C12N15/81
CPCC12N15/81C12N15/52C12N15/65C12N15/74C12N15/75
Inventor 柯尔斯顿·R·本杰明
Owner AMYRIS INC
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