Improving CRISPR Gene Editing with Intentional DNA Mismatches

This patent describes a method to make CRISPR/Cas9 gene editing more effective by intentionally creating small mismatches between the guide RNA and the target DNA sequence.

What it covers

This patent details a genome editing method using the CRISPR/Cas9 system that includes a 'donor nucleic acid molecule' and 'guide RNA'. The core idea is to intentionally create 'one or more mismatched nucleotides' between the target DNA and the guide RNA sequence (Claim 1). This mismatch is designed to increase the efficiency of genome editing (Claim 8). The guide RNA can be a single-chain guide RNA (sgRNA) or a dual RNA (crRNA and tracrRNA) (Claim 2). The donor nucleic acid molecule then causes specific genetic changes, such as substituting, inserting, or deleting nucleotides, or even knocking out or knocking in entire sequences (Claim 5, 6). For example, to correct a specific genetic mutation, a guide RNA with a planned mismatch would direct the Cas9 enzyme to the target DNA, and a donor nucleic acid would provide the correct sequence to be inserted.

What it doesn't cover

• —Does not cover CRISPR/Cas9 genome editing methods that do not involve generating one or more mismatched nucleotides between the target DNA and the guide RNA.
• —Does not cover gene editing systems that do not rely on the CRISPR/Cas9 mechanism, such as zinc finger nucleases or TALENs.
• —Does not cover methods where the guide RNA perfectly matches the target DNA sequence without any intentional mismatches.
• —Does not cover simply delivering CRISPR/Cas9 components to a cell without the specific mismatch strategy described.
• —Does not cover modifications to the Cas9 enzyme itself to improve specificity or reduce off-target effects, unless those modifications are used in conjunction with the mismatch strategy.

The clever bit

The clever bit is intentionally introducing a mismatch between the guide RNA and the target DNA. Instead of aiming for a perfect match, which is the standard approach, this method uses a slight mismatch to surprisingly improve the editing process, specifically by increasing the efficiency of the CRISPR/Cas9 system.

Why it matters

CRISPR/Cas9 is a powerful tool for gene editing, but its efficiency can vary. This patent aims to improve that efficiency, making it more reliable for research and therapeutic applications. Higher efficiency means a greater chance of successfully making the desired genetic change, which is crucial for developing treatments for genetic diseases or creating improved crops. This could reduce the amount of material needed or the number of attempts required to achieve a specific edit.

Real-world examples

• 1.Research labs studying genetic diseases like cystic fibrosis or sickle cell anemia
• 2.Companies developing gene therapies for inherited disorders
• 3.Agricultural biotechnology for creating disease-resistant crops
• 4.Basic science research to understand gene function in cells
• 5.Development of transgenic animals for medical research