Expanding Gene Editing Possibilities with Orthogonal Cas9 Technology

Introduction

Gene editing has emerged as one of the most transformative tools in modern biotechnology, allowing scientists to precisely modify DNA sequences and regulate gene expression. While CRISPR-Cas9 systems have revolutionized genetic engineering, limitations remain when it comes to editing multiple genes simultaneously or avoiding off-target effects. Our patented orthogonal Cas9 proteins offer a significant advancement, enabling RNA-guided gene regulation and editing with enhanced precision and flexibility. By providing orthogonal (non-interfering) Cas9 systems, this technology opens the door to more complex genetic modifications, making it an ideal tool for researchers and companies involved in advanced gene editing and therapeutic development.

Current Challenges in Gene Editing Systems

CRISPR-Cas9 technology has made significant strides in enabling precise gene editing across various organisms. However, traditional CRISPR-Cas9 systems rely on a single type of Cas9 protein, which can limit the ability to simultaneously target and edit multiple genes within the same cell. Additionally, issues with off-target effects—where unintended regions of the genome are modified—remain a concern, particularly in therapeutic applications where precision is critical.

For researchers, scientists, and biotechnology companies aiming to push the boundaries of genetic editing and create multi-gene modifications, there is a growing need for more sophisticated systems that can handle complex genomic tasks without interference or off-target risks.

Orthogonal Cas9 Proteins: Precision and Flexibility

Our orthogonal Cas9 technology introduces a new dimension to gene editing by utilizing multiple Cas9 proteins that operate independently within the same cell. These proteins are orthogonal to each other, meaning they do not interfere with one another’s function, allowing for simultaneous targeting and editing of multiple genes with distinct RNA guides. This system provides a powerful tool for complex genetic modifications, offering researchers the ability to edit multiple genes or regulate several pathways in parallel with greater control and reduced risk of off-target effects.

In therapeutic applications, the use of orthogonal Cas9 proteins allows for more targeted treatments, especially for complex genetic disorders where multiple genes need to be regulated or modified. This technology is also ideal for agricultural biotechnology, where it can be applied to enhance crop traits, improve pest resistance, or accelerate breeding programs through precise gene editing.

Key Benefits

Multi-Target Capability: Enables simultaneous editing or regulation of multiple genes within the same cell, expanding the potential for complex genetic modifications.
Reduced Off-Target Effects: Orthogonal proteins ensure that edits are more specific, minimizing unintended changes in the genome.
Versatile Applications: Applicable in research, therapeutic development, and agricultural biotechnology, making it a powerful tool for various industries.
Enhanced Precision in Genetic Engineering: Provides researchers with greater control over gene editing and regulation, ensuring higher fidelity in outcomes.

Paving the Way for Next-Generation Gene Editing

Licensing this orthogonal Cas9 technology offers companies in biotechnology, pharmaceuticals, and agriculture the opportunity to leverage a powerful and flexible tool for advanced genetic editing. With its ability to target multiple genes simultaneously while minimizing off-target effects, this technology is poised to elevate the possibilities of gene editing and therapeutic development across multiple fields.

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Abstract
Claims

Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

The invention claimed is:

1. A cell in culture comprising

a first foreign nucleic acid encoding two or more guide RNAs complementary to two or more respective target nucleic acids,

a second foreign nucleic acid encoding two or more orthogonal RNA guided nuclease null DNA binding proteins, wherein each of the two or more orthogonal RNA guided nuclease null DNA binding proteins is of a different species and recognizes a corresponding PAM sequence in a target nucleic acid, and

a third foreign nucleic acid encoding two or more transcriptional regulator proteins, wherein the cell is configured to express the two or more guide RNAs, the two or more orthogonal RNA guided nuclease-null DNA binding proteins, and the two or more transcriptional regulator proteins, wherein the cell comprises two or more co-localization complexes with each including a guide RNA, an orthogonal RNA guided nuclease null DNA binding protein, a transcriptional regulator protein and a target nucleic acid.

2. The cell of claim 1 wherein the two or more transcriptional regulator proteins are each fused to an orthogonal RNA guided nuclease null DNA binding protein.

3. The cell of claim 1 wherein the first foreign nucleic acid encoding the two or more guide RNAs further encodes two or more targets of an RNA-binding domain and the third foreign nucleic acid encoding the two or more transcriptional regulator proteins further encodes two or more RNA-binding domains each fused to a transcriptional regulator protein.

4. The cell of claim 1 wherein the cell is a eukaryotic cell.

5. The cell of claim 1 wherein the cell is a yeast cell, a plant cell or an animal cell.

6. The cell of claim 1 wherein each guide RNA includes between about 10 to about 500 nucleotides.

7. The cell of claim 1 wherein each guide RNA includes between about 20 to about 100 nucleotides.

8. The cell of claim 1 wherein a transcriptional regulator protein is a transcriptional activator comprising VP16 or VP64.

9. The cell of claim 1 wherein each of the two or more transcriptional regulator proteins is a transcriptional repressor or activator.

10. The cell of claim 1 wherein each of the two or more guide RNAs is a tracrRNA-crRNA fusion.

11. The cell of claim 1 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

12. The cell of claim 1 wherein a first target nucleic acid is activated and a second target nucleic acid is repressed.

13. The cell of claim 1 wherein a first plurality of target nucleic acids are activated and a second plurality of target nucleic acids are repressed.

14. The cell of claim 1 wherein a first one or more target nucleic acids are activated and a second one or more target nucleic acids are repressed.

15. The cell of claim 1 wherein each of the two or more RNA guided nuclease null DNA binding proteins is a DNA binding protein of a Type II CRISPR system.

16. The cell of claim 1 wherein each of the two or more RNA guided nuclease null DNA binding proteins is an orthogonal nuclease-null Cas9 protein.

17. The cell of claim 1 wherein each of the two or more orthogonal RNA guided nuclease-null DNA binding proteins binds to a corresponding species specific guide RNA bearing matching crRNA and tracrRNA sequences.

Title

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

Inventor(s)

George M. Church, Kevin M. Esvelt, Prashant G. MALI

Assignee(s)

Harvard College

Patent #

10329587

Patent Date

June 25, 2019