Method for targeted modification of algae genomes

A targeted modification and genome technology, applied in the field of genetic material in modified algae cells, can solve problems such as low conversion rate and weak transgene expression

Inactive Publication Date: 2015-12-02
赛莱蒂克斯公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this approach remains difficult due to low transformation rates and weak expression of the transgene, especially in diatoms because their specific silica cell walls contain two separate valves (or shells)

Method used

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  • Method for targeted modification of algae genomes
  • Method for targeted modification of algae genomes
  • Method for targeted modification of algae genomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Increased frequency of targeted mutagenesis at endogenous loci using the PTRI20 meganuclease

[0108] To investigate the ability of a meganuclease to increase the frequency of targeted mutagenesis at an endogenous locus in diatoms, an engineered meganuclease ( known as PTRI20), which was designed to cleave the DNA sequence 5'-GTTTTACGTTGTACGACGTCTAGC-3' (SEQ ID NO: 2). A plasmid encoding a meganuclease and a plasmid encoding a selection gene (Nat1 ) (SEQ ID NO: 3) were co-transformed into diatoms. Mutagenesis rates were measured by deep sequencing on individual clones resulting from transformation.

[0109] Materials and methods

[0110] Culture conditions

[0111] In a Sanyo incubator (model MLR-351) at a constant temperature (20 + / - 0.5°C) on silica-free filtered Guillard's f / 2 medium (40° / °° w / v SigmaSeaSaltsS9883) Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown in medium supplemented with 1X Guillard's f / 2 concentrated seawater solution ...

Embodiment 2

[0125] Example 2: Using a combination of SCTREX2 and PTRI20 meganucleases at diatom endogenous loci High frequency of targeted mutagenesis

[0126] To investigate the ability of the DNA processing enzyme single-stranded TREX2 (SCTREX2) to increase the frequency of targeted mutagenesis induced by meganucleases, an engineered meganuclease encoded by the pCLS17038 plasmid (SEQ ID NO: 1) was used (referred to as PTRI20), which is designed to cleave DNA 5'-GTTTTACGTTGTACGACGTCTAGC-3' (SEQ ID NO: 2). This meganuclease was co-transformed with a plasmid encoding a selection gene (Nat1) (NAT) (SEQ ID NO: 3), and a plasmid encoding a DNA processing enzyme called SCTREX2, encoded by pCLS18296 (SEQ ID NO: 7). Mutagenesis rates were visualized by T7 analysis, measured by deep sequencing on individual clones resulting from transformation.

[0127] Materials and methods

[0128] Phaeodactylum tricornutum Bohlin clone CCMP2561 was cultured according to the method described in Example 1 ...

Embodiment 3

[0144] Example 3: Using a combination of SCTREX2 and PTRI02 meganucleases at diatom endogenous loci High frequency of targeted mutagenesis

[0145]To investigate the ability of the DNA processing enzyme SCTREX2 to increase the frequency of targeted mutagenesis induced by meganucleases, an engineered meganuclease (termed PTRI02) encoded by the pCLS17181 plasmid (SEQ ID NO: 12), which Designed to cleave the DNA sequence 5'-TTTTGACGTCGTACGGTGTCTCCG-3' (SEQ ID NO: 13). A plasmid encoding this meganuclease was co-transformed with a plasmid encoding a selection gene (Nat1) (SEQ ID NO: 3), and a plasmid encoding a DNA processing enzyme, SCTREX2 (encoded by pCLS18296 (SEQ ID NO: 7)). Mutagenesis rates were measured by deep sequencing on individual clones resulting from transformation.

[0146] Materials and methods

[0147] Phaeodactylum tricornutum Bohlin clone CCMP2561 was cultured according to the method described in Example 1 and transformed with M17 tungsten particles (1.1 ...

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Abstract

The invention relates to a method for modifying genetic material in algal cells that includes the use of rare-cutting endonuclease to target specific genomic sequences. In particular, the invention relates to a method for modifying genetic material in algal cells wherein rare-cutting endonuclease, especially a homing endonuclease or a TALE-Nuclease, is expressed over several generations to efficiently modify said target genome sequences.

Description

technical field [0001] The present invention relates to a method for modifying genetic material in algal cells comprising the use of rare-cutting endonucleases to target specific sequences. In particular, the present invention relates to methods for modifying genetic material in algal cells in which rare-cutting endonucleases, especially homing endonucleases or TALE-nucleases, are expressed over several generations to efficiently modify the target sequence. Background technique [0002] Although algae have been used for centuries as a food source for humans, the importance of biotechnological interest in algae, especially microalgae, has only emerged in the last few decades. Applications of algal products range from simple biomass production for food, feed and fuel to valuable products such as cosmetics, pharmaceuticals, dyes, sugar polymers and food supplements. [0003] Several species of algae such as Dunaliella bardawil, Haematococcus pluvialis and Chlorella vulgaris h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22
CPCC12N9/22C12N15/8213C12N9/1241C12N15/52C12Y207/07009C12Y301/00C12Y301/11002C12N1/125C12R2001/89C07K14/195C07K2319/80
Inventor P·迪沙托F·达布西
Owner 赛莱蒂克斯公司
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