The advancements in base editing, a new genome editing approach, have generated a lot of excitement amongst scientists in recent years due to its potential in correcting disease-causing mutations. Base editors combine components from CRISPR systems with other enzymes to allow for the direct, irreversible conversion of single bases within the human genome. Single point mutations are the cause of many detrimental human monogenic disorders, including sickle cell anaemia and cystic fibrosis. As a result, base editors provide an exciting opportunity to make a difference to people’s lives.
Where it began
The ability to precisely and effectively edit human DNA within living cells has been a long-term goal. The recent development of CRISPR-Cas systems was a major step in this direction. The CRISPR-Cas9 system, in particular, generates double-stranded breaks in the DNA at specific loci of interest. Subsequently, cellular DNA repair enzymes fix the breaks, often resulting in the insertion or deletion of bases at the break sites. However, this system often has off-target effects and lacks the ability to precisely fix point mutations. Therefore, attention has turned to increasing the efficiency of gene correction.
The credit for base editing largely belongs to two previous postdoctoral students in David Liu’s laboratory at Harvard University, Alexis Komor and Nicole M. Gaudelli. They developed a pair of molecular machines that could correct the majority of known human disease-related mutations. Their results were later published in Nature and included the first two reported base editors. One paper (2016) described the work led by Komor on the cytosine-to-thymine base editor (CBE). The other (2017) described work led by Gaudelli on the adenine-to-guanine base editor (ABE).
The team at Liu’s laboratory used a catalytically dead Cas9 (dCAS9) that was still able to bind DNA using guide RNA, but not cleave the DNA backbone. They then fused a cytidine deaminase enzyme (rAPOBEC1) to the dCAS9. This enzyme converted cytosine to uracil (first generation, BE1). Next, they added a uracil DNA glycosylase inhibitor (UGI) to prevent the subsequent U:G mismatch from being repaired back to a C:G base pair (second generation, BE2). Finally, to improve efficiency, they restored a specific catalytic residue within the Cas9. This residue was only capable of nicking the non-edited strand. Consequently, this simulated the newly synthesised DNA and produced the desired U:A product. This resulted in a third-generation base editor BE3, APOBEC–XTEN–dCas9(A840H)–UGI. In the 2016 paper, the team reported that when using BE3 up to 37% of the total DNA sequences contained the targeted C>T conversion.
Since then, many research groups have improved these base editors by expanding target scope, improving efficiency and decreasing off-target effects. Researchers have also developed fourth-generation base editors (BE4s). BE4s reduce the undesired C>G or C>A conversions that can happen with BE3s.
The major hurdle in developing ABEs was the lack of any known adenosine deaminase enzymes capable of acting on single-stranded DNA (ssDNA). Liu and team decided to evolve a deoxyadenosine deaminase enzyme that accepted ssDNA. They began with an Escherichia coli transfer RNA adenosine deaminase enzyme, TadA. They then set up large libraries of ecTadA-dCas9 fusions containing mutations in the enzyme portion to detect for the desired activity. From this, they found a specific mutation in the enzyme that was able to make contact with the hydroxyl group of the ribose sugar. They found that mutations at or near TadA D108 allowed TadA to perform adenine deamination on DNA substrates. The evolved TadA enzyme was able to deaminate adenine forming inosine, which is read as a guanine after DNA replication. After further refinement to improve efficiency, the team generated a seventh-generation ABE with approximately 50% efficiency in human cells.
Like CBEs, researchers have made several improvements to ABEs, including targeting flexibility and specificity. Most recently, Ritcher et al. (2020) generated ABE8e which edits ~590-fold faster than the previous ABE7.10. In addition, Guadelli et al. (2020) evolved previous ABEs into 40 new ABE8 variants, which they found to achieve 98-99% target modification in primary T cells. This makes them a promising tool for cell therapy applications.
Several labs have explored the effects of combining both ABE and CBE editing components together. Joung et al. (2020) recently developed a dual-deaminase editor known as SPACE (synchronous programmable adenine and cytosine editor). This editor was able to introduce A>G and C>T substitutions with minimal RNA off-target effects. Furthermore, Li et al. (2020) took a similar approach developing a dual adenine and cytosine base editor – A&C-BEmax. The team fused both deaminases with a Cas9 nickase to achieve C>T and A>G conversions at the same target site. They found that it increased CBE activity and substantially reduced off-target activity.
A recent paper, published in Nature, described a gene editing tool that is able to manipulate mitochondrial DNA (mtDNA) without the use of CRISPR. Editing mtDNA has previously been hindered due to challenges in delivering guide RNA into the mitochondria. Therefore, researchers used an interbacterial toxin (DddA) to generate CRISPR-free, RNA-free DddA-derived cytosine base editors (DdCBEs). These editors catalyse C>T and A>G conversions in human mtDNA. The team found that the efficiency of these editors ranged between 5 to 50%. The team believe that DdCBEs have the potential to model mitochondrial disease mutations and expand our knowledge of mitochondrial biology.
What it offers
Point mutations are the largest group of known pathogenic genetic variants. Collectively, CBEs and ABEs have the potential to fix or reverse up to ~60% of pathogenic point mutations. Since the first development of base editors, researchers have refined and used them to create and correct animal models of human diseases. This includes Duchenne muscular dystrophy, progeria and age-related macular degeneration. Scientists have used base editing to generate mutant mice, rats and rabbits for animal studies.
Liu and his team have previously explored the use of these base editors in several diseases. This includes converting the Alzheimer’s associated allele APOE4 to APOE3r in mouse astrocytes. They have also used BE3 to correct the cancer-associated p53 mutation Tyr163Cys in breast cancer cells. Work by other groups has also shown that direct injection of mRNA encoding base editors into human embryos can generate homozygous mutants at a rate of up to 77%.
Importantly, the precision of base editors has also allowed researchers to make single nucleotide changes that, for example, generate stop codons. This has allowed researchers to test the effects of different gene knockouts across the genome.
The improvement in gene editing tools has transformed the scientific field. It has allowed for advances in scientific research and holds promise for future development of human therapeutics that could potentially cure specific genetic diseases. The increasing precision and versatility of these tools has made the goal of altering desired sequences in any living cell within reach. The rapid evolution of these tools has brought us into a new era that could eventually affect the wider community. Consequently, it is important to engage scientists, governments and other key stakeholders to help guide the next steps. Further improvement of editing capabilities and understanding the consequences of editing our genomes is necessary in ensuring that the benefits of these advancements for society are clear.
Image credit: By CIPhotos – canva.com