How CRISPR Nickases Precisely Edit Genes in Human Cells

This patent describes an engineered CRISPR-Cas system that uses a modified Cas9 protein, called a nickase, to cut only one strand of DNA in a eukaryotic cell, enabling more precise gene editing or altering gene expression.

What it covers

This patent details a method for altering gene expression or editing the genome within a eukaryotic cell, like a human cell. It involves introducing an engineered CRISPR-Cas system that includes a guide RNA and a special Type-II Cas9 protein. Crucially, this Cas9 protein has specific mutations in its cutting part, called the catalytic domain, turning it into a 'nickase' that only cleaves one strand of the DNA molecule (Claim 1). The system also includes at least two nuclear localization signals (NLSs) to ensure it gets into the cell's nucleus where the DNA is located. This single-strand cut can then be repaired by the cell, often with the help of a provided homologous recombination (HR) template, allowing for precise changes to the DNA sequence (Claim 2, 15). For example, this could be used to correct a single faulty letter in a gene that causes a genetic disease.

What it doesn't cover

• —Does not cover CRISPR-Cas systems that create double-strand breaks in DNA, as this system specifically uses a nickase to cleave only one strand.
• —Does not cover gene editing or expression alteration in prokaryotic cells, as the claims specify a 'eukaryotic cell'.
• —Does not cover naturally occurring CRISPR-Cas systems, as the claims require an 'engineered, non-naturally occurring' system.
• —Does not cover Cas9 proteins that lack specific mutations in their catalytic domain, which are necessary to make it a nickase.
• —Does not cover CRISPR-Cas systems that do not include at least two nuclear localization signals (NLSs).

The clever bit

The cleverness lies in modifying the Cas9 protein to become a 'nickase' that only cuts one strand of DNA. This single-strand break is less damaging to the cell than a double-strand break and can be precisely repaired using a template, allowing for highly accurate gene editing with fewer unintended changes.

Why it matters

CRISPR nickases are a vital tool in gene editing because they offer a safer, more precise way to modify DNA compared to traditional Cas9, which creates double-strand breaks. Double-strand breaks can sometimes lead to unwanted, large-scale deletions or insertions in the genome. By only cutting one strand, nickases significantly reduce these off-target effects and enable high-fidelity homology-directed repair, which is crucial for therapeutic applications aiming to correct specific genetic mutations.

Real-world examples

• 1.Gene therapy research for genetic disorders like cystic fibrosis or sickle cell anemia
• 2.Developing disease models in cell lines for drug discovery
• 3.Basic research to understand gene function in mammalian cells
• 4.Precision genome editing for correcting single-nucleotide mutations
• 5.Base editing technologies that build upon nickase capabilities