Precision Control in Gene Expression with RNA-Guided Technology

Introduction

The ability to control gene expression with precision is a fundamental goal in fields like biotechnology, pharmaceuticals, and gene therapy. Traditional methods for regulating gene expression often involve complex processes that lack specificity, resulting in limited control over how genes are activated or silenced. Our patented RNA-guided transcriptional regulation technology offers a powerful, targeted method for influencing gene expression with unprecedented accuracy. This technology opens the door to a wide range of applications, from developing therapies that treat genetic disorders to advancing research in functional genomics and beyond.

The Complexity of Current Gene Regulation Methods

Regulating gene expression is crucial for many therapeutic and research applications, including developing treatments for diseases caused by malfunctioning or overactive genes. However, traditional methods of transcriptional regulation, such as CRISPR-based editing or chemical modulation, come with challenges. They may lead to unintended gene modifications or off-target effects, and they often lack the precision required for finely tuned control of gene activation or suppression.

In both therapeutic and agricultural settings, there is a critical need for tools that can modulate gene expression more precisely without causing permanent alterations to the genetic code. This is particularly important in developing personalized medicine, where the ability to adjust gene activity for individual patients can significantly impact treatment success.

A Game-Changing Approach with RNA-Guided Regulation

Our patented RNA-guided transcriptional regulation technology introduces a groundbreaking method for controlling gene expression using RNA molecules. By guiding transcription factors to specific locations in the genome, this system enables precise activation or suppression of genes without altering the DNA sequence itself. This approach provides an elegant solution for modulating gene activity, offering more control, fewer side effects, and the ability to reverse or fine-tune gene expression as needed.

This technology is versatile and can be applied in a variety of settings, including gene therapy, where it offers a safe and reversible way to regulate disease-related genes. In pharmaceutical development, it can accelerate the discovery of gene-based therapies by enabling more accurate control over experimental conditions. Additionally, in agricultural biotechnology, this system can be used to regulate plant traits without permanent genetic modifications, offering a promising tool for sustainable crop development.

Key Benefits

Precise Gene Regulation: Enables highly specific control over gene activation or suppression without altering the underlying DNA.
Reversible and Adjustable: Provides the flexibility to fine-tune or reverse gene expression based on therapeutic or research needs.
Wide Application: Suitable for use in gene therapy, pharmaceutical research, and agricultural biotechnology.
Fewer Off-Target Effects: Minimizes unintended changes to the genome, ensuring safer applications in human therapies and research.

Unlocking New Possibilities in Gene Therapy and Research

Licensing this RNA-guided transcriptional regulation technology offers biotechnology and pharmaceutical companies a powerful tool for advancing precision medicine and gene-based therapies. With its ability to modulate gene expression safely and accurately, this innovation paves the way for new treatments, groundbreaking research, and advancements in agricultural biotechnology.

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Abstract
Claims

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

1. A method of inserting a donor nucleic acid sequence into a cell using homologous recombination comprising

providing to the cell two guide RNAs with each guide RNA having a spacer sequence, a tracr mate sequence and a tracr sequence, and with a portion of the tracr sequence being hybridized to the tracr mate sequence and with the tracr mate sequence and the tracr sequence being linked by a linker nucleic acid sequence and with each spacer sequence being complementary to an adjacent site in a DNA target nucleic acid,

providing to the cell a donor nucleic acid sequence,

providing to the cell a Cas9 protein nickase, and

wherein each of the two guide RNAs co-localize with the Cas9 protein nickase to the DNA target nucleic acid resulting in an offset nick with 5′-overhangs, and

wherein the donor nucleic acid sequence is inserted into the target nucleic acid at the offset nick using homologous recombination.

2. The method of claim 1

wherein the two guide RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid encoding the two guide RNAs,

wherein the Cas9 protein nickase is provided to the cell by introducing into the cell a second foreign nucleic acid encoding the Cas9 protein, and

wherein the two guide RNAs and the Cas9 protein nickase are expressed.

3–11. (canceled)

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

13–24. (canceled)

25. The method of claim 1 wherein the tracr sequence is between 90 to 500 nucleotides.

26. The method of claim 1 wherein the tracr sequence is between 90 to 200 nucleotides.

27. The method of claim 1 wherein the tracr sequence is between 100 to 200 nucleotides.

Title

RNA-Guided Transcriptional Regulation

Inventor(s)

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

Assignee(s)

Harvard College

Patent #

20200024618

Patent Date

January 23, 2020