New genome editing tools called multiplexed orthogonal base editors (MOBEs) install multiple point mutations at once, according to researchers from the University of California San Diego (UCSD) who developed them. MOBEs use RNA structures called aptamers—small RNA loops that bind to specific proteins—to recruit base-modifying enzymes to specific genomic locations enabling simultaneous editing of multiple sites with high efficiency and a lower incidence of crosstalk.
The researchers say this is the first time someone used aptamers to recruit ABEs (adenosine base editors) in combination with CBEs (cytosine base editors) in an orthogonal pattern to make the MOBEs.
“Most variants that are identified are unclassified clinically, so we don’t even know if they’re pathogenic or benign,” said Quinn T. Cowan, a recent PhD graduate from UCSD’s Department of Chemistry and Biochemistry and first author on the paper. “Our goal was to make a tool that can be used in disease modeling by installing multiple variants in a controlled laboratory setting where they can be studied further.”
Their work appears in Nature Biotechnology, May 21, 2024. It was led by Alexis C. Komor, PhD, of the UCSD Department of Chemistry and Biochemistry.
Current methods to model or correct mutations in live cells are inefficient, these researchers say, especially when multiplexing—installing multiple point mutations simultaneously across the genome. Komor’s team was especially interested in comparing genomes that differ at a single letter change in the DNA.
One issue with using the genome in disease modeling is the sheer number of possible variations. Scientists trying to determine which genetic mutations are responsible for heart disease, they could decode the genomes of a cohort that all had heart disease but the number of variations between any two people makes it very hard to determine which combination of variations causes the disease.
The traditional gene-editing tool CRISPR-Cas9, uses a guide RNA, which acts like a GPS signal that goes straight to the genomic location targeted for editing. Cas9 is the DNA-binding enzyme that cuts both strands of the DNA, making a complete break, but double-stranded breaks can be toxic to cells. This kind of gene-editing can also lead to indels— random insertions and deletions—where the cell is not able to perfectly repair itself. Editing multiple genes in CRISPR-Cas9 multiplies the risks.
Instead of CRISPR, Komor’s lab uses a base-editing technique she developed, which makes a chemical change to the DNA, although only one type of edit (C to T or A to G, for example) can be made at a time. So rather than scissors that cut out an entire section at once, this version of base-editing erases and replaces one letter at a time. It is slower, but the researchers say it is more efficient and less harmful to cells.
As the researchers write, they engineered, “ Both adenine BEs [base editors] and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer–coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs.” The team generated four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies.
The differences are stark: they found that when CBE and ABE were given together not using MOBE, crosstalk occurred up to 30% of the time. With MOBE, crosstalk was less than 5%, while achieving 30% conversion efficiency of the desired base changes.
