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Construction and application of marine nannochloropsis oculata transcriptional activation CRISPRa system

A marine Nannochloropsis, transcription activation technology, applied in applications, microorganism-based methods, microorganisms, etc., can solve the problem of slow establishment of gene editing methods, improve multi-stress resistance, increase oil production, and increase biomass. Yield effect

Active Publication Date: 2022-05-13
HAINAN NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this technology is used in plants, especially in microalgae. Due to the slow establishment of gene editing methods, and the understanding of the transcriptional activation complex system and whether other commonly used transcriptional activation domains can be applied in microalgae is still unclear. Transcriptional activation systems are still rarely established in microalgae

Method used

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  • Construction and application of marine nannochloropsis oculata transcriptional activation CRISPRa system
  • Construction and application of marine nannochloropsis oculata transcriptional activation CRISPRa system
  • Construction and application of marine nannochloropsis oculata transcriptional activation CRISPRa system

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Embodiment

[0046] Realize the construction of the targeted transcriptional activation system of the endogenous gene methyltransferase gene (g1248) in Nannochloropsis:

[0047] 1. Artificial synthesis can express dCas9 protein (codon optimization, adjusted by GC content to be suitable for expression in Nannochloropsis), VP64 domain (codon optimization), SunTag sequence (codon optimization), hygromycin resistance Gene (codon-optimized), transcriptional activation vector (CRISPR / dCas9-Suntag-VP64) for nuclear localization signal SLV (codon-optimized):

[0048] 1) Backbone vector: using the known marine Nannochloropsis genome (NCBI: ASM187094v1 ) sequence, with the Nannochloropsis gene editing vector (pNOC-ARS-CRISPR) as the backbone, the backbone vector contains endogenous promoters (Ribi and LDSP) and LDSP terminators, which can drive the expression of Cas9 protein expression Edited expression vector.

[0049] 2) Synthesis of dCas9 protein: inactivate the Cas9 protein or replace the ori...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a marine nannochloropsis oculata transcriptional activation CRISPRa system and a construction method and application thereof. The system vector contains a Cas9 inactivated protein, a transcriptional activation effect protein VP64, a resistance selection marker gene, at least one gRNA scaffold sequence and a specific sequence for enhancing transcriptional activation (SunTag sequence). The invention also provides the application of the CRISPRa vector for the transcription activation of the nannochloropsis oculata gene in gene transformation. The invention provides a new model and a feasible method for gene function research of nannochloropsis oculata.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a CRISPRa system for transcriptional activation of marine Nannochloropsis and its construction method and application. Background technique [0002] Marine Pseudochloropsis Nannochloropsis is a single-celled photosynthetic microalgae widely distributed in oceans, lakes, swamps and other waters in different water environments. Due to its high oil content, good environmental adaptability and suitability for flue gas cultivation, it is favored in the fields of microalgae bioenergy, microalgae functional food, microalgae feed, etc., and is the "blue granary" One of the important biological resources. With the rise of synthetic biology in recent years, Nannochloropsis has emerged as one of the photosynthetic chassis cells of a new generation of synthetic biology, and has developed to an equally important position as model algae strains such as Chlamydomonas reinhardti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/67C12N15/55C12N1/13C12R1/89
CPCC12N15/8218C12N15/67C12N9/22Y02A50/30
Inventor 魏力
Owner HAINAN NORMAL UNIV
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