CRISPR Tools for Engineering Fungal Genomes

This patent describes a specific genetic tool, called an expression vector, designed to efficiently edit the DNA of fungi using the CRISPR-Cas9 system, which is useful for industrial applications like making biofuels or enzymes.

What it covers

This patent provides a special genetic package, or "expression vector," that helps scientists edit the DNA of fungi. This vector contains several key parts: an "RNA polymerase III promoter" to start the gene editing process, a "ribozyme" that acts like tiny scissors, a "CRISPR-Cas9 single guide RNA" (sgRNA) to direct the editing to a specific DNA spot, and an "RNA polymerase III terminator" to stop the process (Claim 1). A unique feature is that the ribozyme is placed just before the sgRNA. When this vector is put into a fungal cell, like yeast (Claim 13), it allows the cell to produce the necessary components to precisely cut and modify its own DNA (Claim 20). For example, this could be used to introduce a new gene, such as a "cellodextrin transporter" (Claim 23), into a yeast cell to help it break down plant material more efficiently for biofuel production.

What it doesn't cover

• —Does not cover genome editing in non-fungal organisms, such as human, animal, or plant cells (Claims 13-19).
• —Does not cover CRISPR-Cas systems that do not use a Cas9 protein or a single guide RNA (Claim 1).
• —Does not cover expression vectors where the ribozyme is not positioned immediately before (5' to) the single guide RNA (Claim 1).
• —Does not cover gene editing methods that do not rely on an RNA polymerase III promoter and terminator (Claim 1).

The clever bit

The clever part is the specific design of the expression vector, particularly the arrangement of the ribozyme positioned 5' to the CRISPR-Cas9 single guide RNA, combined with the use of RNA polymerase III elements. This specific setup ensures that the guide RNA is correctly processed and expressed in fungal cells, making the CRISPR-Cas9 editing system work more reliably and efficiently in these organisms.

Why it matters

Fungi, especially yeast, are crucial for many industrial processes, from making bread and beer to producing biofuels, enzymes, and medicines. This patent provides a more effective and precise way to engineer the genomes of these "industrial strain" fungal cells (Claim 14). By making it easier to modify fungal DNA, scientists can improve how these organisms produce valuable products, potentially leading to more efficient and cost-effective manufacturing in various industries.

Real-world examples

• 1.Yeast strains (Saccharomyces cerevisiae) used in biofuel production
• 2.Fungi engineered to produce industrial enzymes
• 3.Yeast used in the fermentation of food and beverages
• 4.Fungal cells modified to produce pharmaceuticals