How to Precisely Edit Genes Using Retron-Guide RNA Cassettes

This patent describes a method for highly efficient and precise genome editing using a retron-guide RNA cassette to deliver large pieces of donor DNA into a cell's genetic material.

What it covers

This invention provides a system for editing genes with high precision and efficiency. It uses a special genetic package called a "retron-guide RNA cassette" (Claim 1). This cassette contains a "retron," which is a genetic element with specific parts like an "msr locus," an "msd locus" (where the new "donor DNA sequence" is placed), and inverted repeat sequences (Claim 1). Importantly, the donor DNA sequence, which carries the genetic changes to be inserted, is quite long, ranging from 500 to 10,000 nucleotides (Claim 1). The cassette also includes a "guide RNA (gRNA) coding region" (Claim 1). When introduced into a cell, the retron creates an RNA molecule that can make many copies of single-stranded DNA (msDNA) using a "reverse transcriptase (RT)" (Claim 4, 5). This msDNA, containing the donor DNA, then precisely inserts into the target gene location in the cell's genome, guided by the gRNA and matching "homology arms" (Claim 13) on the donor DNA. For example, this system could be used to correct a large faulty gene sequence responsible for a genetic disorder.

What it doesn't cover

• —Does not cover genome editing systems that do not use a retron to deliver the donor DNA.
• —Does not cover methods where the donor DNA sequence is shorter than 500 nucleotides in length.
• —Does not cover gene editing techniques that do not involve a guide RNA molecule.
• —Does not cover systems that deliver donor DNA without generating multicopy single-stranded DNA (msDNA) via self-priming reverse transcription.
• —Does not cover gene editing where the donor DNA lacks homology arms for precise integration at a nuclease cleavage site.

The clever bit

The novelty lies in using retrons, which are bacterial genetic elements, to generate multiple copies of a large donor DNA sequence inside a cell. This allows for highly efficient delivery and integration of significant genetic material, overcoming limitations of other gene editing tools that struggle with large inserts or high efficiency.

Why it matters

This technology offers a way to insert much larger pieces of DNA into a genome than many existing methods, which is crucial for correcting complex genetic errors or adding new functions. The high efficiency promised by retrons could make gene editing therapies more effective for treating a wider range of genetic diseases. It could also accelerate research by allowing scientists to more easily modify genes in laboratory settings.

Real-world examples

• 1.Correcting large gene mutations in genetic diseases like cystic fibrosis or Duchenne muscular dystrophy.
• 2.Inserting therapeutic genes into cells for gene therapy applications.
• 3.Developing new disease models in laboratory animals by precisely modifying their genomes.
• 4.High-throughput screening platforms for drug discovery by creating diverse cell lines with specific genetic changes.