Targeting repetitive sequences for gene editing

Regions of the genome with repetitive sequences are difficult to selectively edit because it’s hard to control which of the repeated sections will be edited. Ribosomes, the molecular machines that synthesize proteins in cells, are examples of complexes that contain such repetitive sequences. Researchers would like to engineer ribosomes to accept substrates other than the collection of 20 amino acids they usually use as protein building blocks, but doing so would require altering repetitive genomic sequences.

Farren J. Isaacs of Yale University and coworkers have now developed a strategy for controlling which copies of repeated sequences get edited (Nat. Commun. 2022, DOI: 10.1038/s41467-021-27836-x). They demonstrated the approach, which they call filtered editing, with both CRISPR and another method called multiplex automated genomic engineering (MAGE).

The researchers inserted a DNA sequence that differentiates one repetitive section from the others without interfering with the section’s function. The inserted DNA sequence encodes a “self-splicing intron.” The intron provides a unique address for genome-editing machinery to home in on so it can carry out its editing function on the selected section. After the edited DNA sequence is transcribed as RNA, the intron splices itself out of the RNA and closes the gap.

Isaacs and coworkers used the approach to introduce changes to ribosomes in Escherichia coli, specifically, to evolve antibiotic-resistant ribosomes. They also showed they could make changes to orthogonal ribosomes—ones that can incorporate unnatural amino acids or other substrates without interfering with native ribosomes—in bacterial cells without making changes to the native ribosomes. They made edits at locations in the small and large subunits of the orthogonal ribosomes simultaneously, including the messenger RNA binding site in the small subunit and the catalytic center and exit tunnel in the large subunit.

The method should make it easier to engineer ribosomes, according to Michael C. Jewett of Northwestern University, who is working on engineering orthogonal ribosomes to accept a variety of substrates. “Using self-splicing introns as a clever innovation, ribosome engineering efforts can shift to the genome,” he writes in an email. “I expect this method to be applied to engineer the ribosome extensively to work with exotic substrates beyond those in nature, enabling sustainable polymers that benefit society.”

Isaacs wants to evolve multiple parts of the translation system, including transfer RNAs and tRNA synthetases. “You need to be able to fundamentally reengineer entire organisms from the genome level through the translation system and the ribosome to enable the biosynthesis of new biopolymers or materials,” he says.

Isaacs also plans to extend the approach to other organisms. “The introduction of self-splicing to create unique genetic addresses is easily adaptable to higher order eukaryotes,” he says. “I actually think there’s a nice blueprint here for gaining access to repetitive elements in diverse organisms using whatever genome editing tool the researcher prefers.”