Highly Efficient Bacterial Gene Editing Using Guide RNA and Reverse Transcriptase

This patent describes a system for precisely editing the DNA of bacterial cells with very high success rates, using a combination of guide RNA, reverse transcriptase, and specific DNA sequences.

What it covers

This patent describes an engineered nucleic acid construct designed for highly efficient gene editing in bacterial cells. The construct includes three main parts: a nucleotide sequence encoding a guide RNA that targets an exonuclease (like RecJ, XonA, or ExoX, as per claim 5), a sequence for a modified single-stranded msrRNA and msdDNA containing a specific targeting sequence flanked by inverted repeats (claim 1b), and a sequence for a reverse transcriptase protein (claim 1c). When delivered to a bacterial cell, this system works to modify specific target nucleotide sequences, such as an undesired allele of a gene (claim 20), by using the guide RNA to disable exonucleases and the reverse transcriptase to help incorporate the new DNA, leading to nearly 100% recombination efficiency.

What it doesn't cover

• —Does not cover gene editing systems that do not include a guide RNA specifically targeting an exonuclease.
• —Does not cover gene editing in eukaryotic cells, as the claims specify bacterial cells.
• —Does not cover methods that lack a reverse transcriptase protein as a component of the engineered construct.
• —Does not cover systems where the single-stranded msrRNA and msdDNA targeting sequence is not flanked by inverted repeat sequences.
• —Does not cover gene editing approaches that rely solely on CRISPR-Cas9 without the additional components like exonuclease targeting and reverse transcriptase.

The clever bit

The clever bit is the specific combination of components designed to achieve extremely high editing efficiency. By having a guide RNA target and disable exonucleases, the system prevents the cell from chewing up the new DNA, while the reverse transcriptase helps incorporate the desired changes, ensuring that almost every targeted bacterial cell is successfully modified.

Why it matters

Achieving nearly 100% recombination efficiency in bacterial gene editing is a significant advancement. This high success rate makes it much easier and faster to engineer bacteria for various purposes, from producing medicines and biofuels to developing new probiotics or studying bacterial diseases. It reduces the time and effort needed to isolate correctly modified cells, accelerating research and industrial applications.

Real-world examples

• 1.Engineering bacteria for industrial chemical production
• 2.Developing bacterial strains for bioremediation
• 3.Creating designer probiotics for gut health
• 4.Research tools for studying bacterial genetics and disease mechanisms
• 5.Optimizing bacterial strains for vaccine production