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

A target nucleic acid, nucleic acid technology, applied in the field of nucleic acid, composition and method for excising target nucleic acid, and can solve the problems of low frequency of excision events and the like

Active Publication Date: 2015-11-25
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 to find ways to select for rare host cells undergoing excision events by growth

Method used

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

Examples

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Comparison scheme
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Embodiment 1

[0125] 6.1 Example 1: Construction of x-marker constructs

[0126] The compositions and methods described herein were used to prepare and identify a series of excisable selectable markers for Saccharomyces cerevisiae, referred to herein as "x-markers". The first generation of labeling uses I-SceI endonuclease, and the parameters of the DNA constructs detected are given in figure 1 , parameters included: (1) different lengths of the forward repeat flanks of the endonuclease sites and (2) a comparison of one and two endonuclease sites. The second generation of markers applies the results of the first generation of assays, which broaden the application of the I-SceI endonuclease. The third-generation x-label has proven effective and can be extended to other endonucleases listed in Table 1 below.

[0127] Table 1: Endonuclease recognition and cleavage sites.

[0128]

[0129] Reagents used in the experiments are described below and restriction enzymes were from New England B...

Embodiment 2

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

[0192] This example demonstrates that the x-marker construct described in Example 1 is capable of mediating excision of the selectable marker from the chromosomal DNA of the host cell. As described below, the constructs are transformed into cells, cells are plated into media selective for the x-marker, and correct integration is confirmed by colony PCR; such strains are treated in the same manner as any other prepared using standard markers The strains are the same, and the x mark can exist stably. Third, when excision of the x-marker is desired, transform the strain with a single-copy (CEN.ARS) plasmid containing its own marker and a homing endonuclease gene expression construct, as described in the following sections. Strains were tested for loss of the x-marker after several generations of selection for the presence of the plasmid and growth under conditions that induced expression of the homing endon...

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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 priority to US Provisional Application No. 61 / 378,350, filed August 30, 2010, the entire contents of which are incorporated herein by reference. 1. Technical field [0002] The nucleic acids, compositions, and methods provided herein relate generally to 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 technology to excise target nucleic acid from the host cell genome or episome. However, previous methods for removing target nucleic acids have been constrained and limited in their application. For example, removal using a site-specific recombinase approach would leave deleterious specific recombinase binding sites that create potential genomic instability in the host cell. Other methods produce excision events less frequently, and ...

Claims

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Application Information

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