Current methods for modeling or correcting mutations in live cells are not very effective, especially when it comes to multiplexing – making multiple point mutations simultaneously across the genome. However, researchers have created new, effective genome editing tools known as multiplexed orthogonal base editors (MOBEs) that can be used to simultaneously make multiple point mutations.
The human genome is made up of approximately 3 billion base pairs, and despite this large number, humans are 99.6% identical in terms of their genetic makeup. The remaining 0.4% accounts for the differences between individuals. Specific combinations of mutations in these base pairs provide important information about the differences and similarities between people.Complex health issues, such as heart disease and neurodegenerative diseases like schizophrenia, have various causes. Current techniques for modeling or repairing mutations in living cells are not very effective, particularly when it comes to multiplexing – making multiple point mutations simultaneously across the entire genome. A team of researchers from the University of California San Diego has created new genome editing tools known as multiplexed orthogonal base editors (MOBEs) to efficiently introduce multiple point mutations at the same time. This work, led by Assistant Professor of Chemistry and Biochemistry Alexis Komor’s lab, is featured in Nature Biotechnology.
Komor’s team focused on comparing genomes with single letter changes in the DNA, also known as C (cytosine), T (thymine), G (guanine), A (adenosine) bases. These single nucleotide variants (SNVs) or single point mutations can result in 4-5 million variants in a person. Some of these variants are harmless, while others can be harmful, and it’s often a combination of variants that leads to disease.
Using the genome in disease modeling presents a challenge due to the large number of possible variations. Scientists need to identify which variants contribute to specific diseases.
If specific genetic mutations were found to be the cause of heart disease, researchers could analyze the genetic makeup of a group of individuals all affected by heart disease. However, the vast number of differences between any two people makes it incredibly challenging to determine which specific combination of variations contributes to the disease.
“The interpretation of genetic variations poses a significant challenge. In fact, the majority of identified variants are not clinically classified, so we lack understanding of whether they are disease-causing or harmless,” explained Quinn T. Cowan, a recent Ph.D. graduate from the university’s Department of Chemistry and Biochemistry and the primary author of the study. “Our objective was to develop a tool specifically designed for disease assessment.”
An advancement in gene-editing
In order to comprehend the purpose behind the development of MOBEs, it is important to recognize the limitations of the conventional gene-editing tool CRISPR-Cas9. CRISPR-Cas9 utilizes a guide RNA, which functions as a GPS signal directing it to the specific genomic location that needs to be edited. Cas9 is the DNA-binding enzyme that cuts both strands of the DNA, resulting in a complete break.
Although the process is relatively simple, double-stranded breaks can be harmful to cells. This type of gene editing can be toxic, which is why MOBEs were created. These MOBEs allow for the study of multiple variants in a controlled laboratory setting.
The process of editing using CRISPR-Cas9 can result in indels, which are random insertions and deletions in the DNA where the cell is unable to repair itself perfectly. Editing multiple genes using CRISPR-Cas9 increases the potential risks associated with this technique. Instead of using CRISPR, the lab of Komor utilizes a base-editing method that she developed. This technique induces a chemical alteration to the DNA, allowing for the modification of a single letter at a time, such as changing a C to a T or an A to a G. Unlike the CRISPR method, which cuts out an entire section of DNA at once, base-editing erases and replaces one letter at a time. Although this approach is slower, it is more efficient and poses less harm to the cells. By applying two or more base editors simultaneously, the potential exists for even greater efficiency.The ability to make precise changes to the DNA (such as changing a C to T at one location, and an A to G at another location in the genome) allows for better understanding and modeling of polygenic diseases – those caused by more than one genetic variant. However, until now, there hasn’t been a technology that could do this efficiently without guide RNA ”crosstalk,” which occurs when base editors make unwanted changes.
Cowan’s MOBEs use RNA structures known as 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.
This system is innovative and unique.This is the first instance where aptamers were utilized to combine ABEs (adenosine base editors) and CBEs (cytosine base editors) in an independent manner to create MOBEs. The differences are significant: when CBE and ABE are administered together without using MOBE, crosstalk occurs up to 30% of the time. With MOBE, crosstalk is less than 5%, while achieving 30% conversion efficiency of the desired base changes. The research served as a proof of concept to assess the potential of the MOBE system, which has been provisionally patented. To further test them, the team conducted various case studies with real-life applications.l diseases, such as Kallmann syndrome, a rare hormonal disorder. Their studies showed that MOBE systems could effectively edit specific cell lines related to certain polygenic diseases.
“We are currently in the process of making the plasmids available on AddGene so that anyone can access them freely. Our goal is for other scientists to use the MOBEs to simulate genetic diseases, understand their symptoms, and ultimately develop effective treatments,” Cowan explained.
This study received funding from the National Institutes of Health (1R35GM138317, T32 GM008326, and T32 GM112584) and the Research Corporation for Science Advancement (28385).
