Target gene Sequence Specific Nucleases Plant Genome Editing

Introduction

It is helpful to boost the efficiency of crop oriented genetic programming by using gene editing technique to delete unfavorable gene and improve the target characters accurately. Benifit from its convenient, accurate and multi-targets gene editing function, CRISPR-Cas9 has successfully applied in plant genes to improve the phenotypic characteristics against insect pests and drought. It is expected to be widely used in precise genetic improvement of crops. Precise plant gene editing has been listed as one of the ten major scientific and technological breakthroughs in 2016. The targeted gene engineering based on CRISPR-Cas9 has gradually replaced traditional plant breeding and genetically modified organism (GMO) methods to improve crop characteristics to ensure thesustainable production of crops.

The emergence of sequence-specific nucleases that enable genome editing is revolutionizing basic and applied biology. Since the introduction of CRISPR-Cas9, genome editing has become widely used in transformable plants for characterizing gene function and improving traits, mainly by inducing mutations through non-homologous end joining of double-stranded breaks generated by CRISPR-Cas9. However, it would be highly desirable to perform precision gene editing in plants.

Flow chart

1. SgRNA design and construction

Generally, sgRNA are driven by U3 or U6 small nuclear RNA gene promoters. To insert a few sgRNA cassettes in a single binary vector is recommended when multiple genome targets are required in plant.

2. Delivery of Cas9 and sgRNA into plant cells

The Agrobacterium-mediated transformation is the most common way to introduce the Cas9/sgRNA expression cassettes into plant cells. The other methods are available if request.

3. Analysis of targeted mutations

Endonucleases (e.g. T7E1 enzyme)

Sanger Sequencing

4. Delivery

Advantages

• CELLFREE provides complete solutions from sgRNA design, Cas9 codon optimization, sgRNA and Cas9 synthesis and constructionto activity detection.
• CELLFREE provides efficient and accurate designs for multiple species.
• CELLFREE Optimize Cas9 protein to ensure its efficient translation in plant cells.