Many human traits are influenced by multiple genes. Human genes can also carry slight variations in their gene sequences, which can have a wide range of effects. In some complex traits, those small changes in multiple genes can have an additive impact that might increase or decrease a person's risk of disease. But studying those combinations of genetic variations can be extremely challenging. Scientists have now developed a new technique for studying how small, single nucleotide variations or so-called point mutations can combine to have an influence on biology. This new method utilizes multiplexed orthogonal base editors (MOBEs), and can be used in cell lines. The work has been reported in Nature Biotechnology.
There are four letters or bases in the human genome: A (adenosine), G (guanine), C (cytosine), and T (thymine), and there are about 6 billion bases in our DNA. One person might carry about 5 million variants in their genome, which help make each one of us unique, but can also carry an increased (or decreased) risk of some health condition. But the number of possible combinations of variants makes studying them quite daunting. Some of those variants may have little or no effect either.
"There is a problem interpreting genetic variants. In fact, most variants that are identified are unclassified clinically, so we don't even know if they're pathogenic or benign," explained first study author Quinn T. Cowan, PhD. "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."
Although the CRISPR-Cas9 gene editing tool is very popular, introducing a single genetic change requires a number of reagents, including one which is an RNA molecule that 'guides' the gene-editing machinery to the right place in the genome, where a cut is made. When researchers want to make multiple edits, however, it becomes increasingly hard for the cell to deal with all of the cuts being made in the genome; the chances that unwanted cuts will be made and undesirable changes will happen in other parts of the genome are increased. It can also be very hard to know when these off-target effects have occurred.
In this new approach, a base editor is used instead. With base editors, a single base like C can be changed to T or A to G, for example. This is a slower process compared to CRISPR-Cas9, but there is less harm to the genome and less danger to cells.
Two or more base editors can also be applied to cells at once, so complex traits become easier to model. In this work, the scientists made those base editors more specific and less likely to make unwanted changes by adding RNA molecules called aptamers.
This is the first time aptamers have been combined with molecules called adenosine base editors (ABEs) and cytosine base editors (CBEs) to create MOBEs.
The researchers demonstrated the efficiency of the MOBE system by modeling several complex diseases with known genetic changes in cell lines.
"We're in the process of putting the plasmids up on Addgene so anyone can freely access them. Our hope is that other researchers will use the MOBEs to model genetic diseases, learn how they manifest and then hopefully create effective therapies," stated Cowan.
Sources: University of California - San Diego, Nature Biotechnology
