What is single-base editing?

Gene editing uses enzymes to make precise changes to the DNA sequence of a gene. Editing can correct genetic defects or introduce new traits into an organism. There are several different techniques for gene editing, including CRISPR/Cas9, which uses a programmable enzyme called Cas9 to cut double-stranded DNA at a specific location. The desired nucleotides can be inserted, deleted, or modified at that location. In a previous article, I described how CRISPR/Cas9 uses clustered regularly interspaced short palindromic repeats (CRISPR) of DNA and an enzyme (Cas9) that cuts or hydrolyzes DNA. This is also called simply CRISPR. It is being used to create new foods ¹. CRISPR may become an important part of the fourth industrial revolution. It is being used by scientists to cut and paste almost any desired DNA sequence. Faulty genes can be cut out and replaced with the desired, functioning gene, or to insert new genes that give the recipient better qualities. It has been used to make genetically modified bacteria (Streptococcus thermophilus) that are used to make yogurt and cheeses ².

One potential use of gene editing is in the development of new crops and plants with improved traits. For example, researchers have used CRISPR/Cas9 to create rice plants that are more resistant to pests and diseases, which could help increase crop yields and reduce the need for chemical pesticides. Gene editing has also been used to produce plants with improved nutritional content, such as rice with higher levels of vitamin A, which could help address malnutrition in developing countries.

Another potential application of gene editing is in the production of new pharmaceuticals. For example, researchers have used CRISPR/Cas9 to produce yeast strains that can synthesize artemisinin, an anti-malarial drug, more efficiently than current methods. Gene editing could also be used to create microorganisms that can produce other valuable pharmaceuticals, such as proteins for use in biotechnology and medicine.

Gene editing has the potential to revolutionize the field of medicine by allowing researchers to precisely target and correct genetic mutations that cause diseases. For example, researchers have used CRISPR/Cas9 to correct a genetic mutation that causes sickle cell anemia in human cells in a laboratory setting. In the future, gene editing may be used to treat a wide range of genetic diseases, including cystic fibrosis, muscular dystrophy, and Huntington's disease.

Gene editing could also have important applications in environmental protection. For example, researchers have used CRISPR/Cas9 to create genetically modified trees that are more resistant to pests and diseases, which could help prevent deforestation. Gene editing could also be used to create microorganisms that can break down environmental pollutants, such as oil spills and toxic chemicals, more efficiently than current methods.

Gene editing has the potential to revolutionize many different industries, including agriculture, pharmaceuticals, and environmental protection. While there are still many challenges to overcome, such as ensuring the safety and efficacy of gene-edited organisms, the potential applications of gene editing are vast and varied, and this technology has the potential to have a major impact on society in the coming decades.

While CRISPR/Cas9 and other gene editing techniques can be used to make precise changes to the DNA sequence of a gene, they are not capable of cutting and pasting any desired sequence. There are limitations to what can be achieved with these techniques, and the specific changes that can be made depending on the specific system being used and the specific sequence being targeted.

CRISPR has been used to produce yeasts that can consume plant matter and excrete ethanol, which could help end our addiction to fossil fuels. Perhaps the most important example is the production of improved microalgae. They are single-cell photosynthetic organisms that could be a sustainable and environmentally friendly sources of food and fuel. The microalgae called Spirulina (Arthrospira platensis) were recognized as the best food for the future at the United Nations World Food Conference in 1974. During the 60th session of the United Nations General Assembly, a resolution was issued stating that Spirulina can be used to fight hunger and malnutrition and help achieve sustainable development. Chlorella and Spirulina are sold in health food stores because they are some of the most nutritious foods known. They contain not only protein, polysaccharides, sterols and lipids, but also many vitamins, carotenoids and polyphenols.

CRISPR/Cas9 and other means of editing genes are also being used in medicine, environmental protection, industrial biotechnology and basic research. Potentially, they can be used to cure genetic diseases by correcting mutations that cause the disease. This could be especially useful for diseases that have no cure or are caused by multiple genetic mutations. Gene editing may also help restore populations of endangered species and create genetically modified organisms that can help clean up environmental pollutants. It can be used to create microorganisms that will be able to produce valuable chemicals, fuels, or other products more efficiently than current methods. Gene editing is also being used to study the function of specific genes and understand how they affect various processes in the human body or in other organisms. This knowledge can help researchers develop new treatments and therapies for a wide range of diseases and conditions.

While it is possible that gene editing could be used to help restore populations of endangered species, it is important to note that the use of genetically modified organisms (GMOs) to clean up environmental pollutants is still an area of active research and development, and it is not yet clear how effective this approach will be in practice.

Nonmodel microbes that are derived from complex environments have evolved to utilize cheaper and more environmentally friendly sources of carbon than model microbes3. Some have versatile physiology and metabolic capabilities that may be used to produce biofuel, as well as new antibiotics that could ease the problems caused by multi-drug-resistant pathogenic bacteria. They are also able to tolerate extreme industrial processing environments, such as low pH, high salt, and high temperatures; therefore, nonmodel microbes have become one of the hot spots in metabolic engineering. However, the lack of facile gene editing tools hinders the genetic characterizations and modifications of nonmodel microbes. Moreover, understanding and mitigating drug-resistance mechanisms require gene engineering approaches to aid in targeting and editing pathogenic microbial genomes ³.

Gene editing technology has improved to where it can modify a single nucleotide base from DNA. This is called base editing ⁴. Base editors use facets of CRISPR–Cas9 by combining enzymes that modify DNA with a catalytically inactive form of Cas9. With base editors, labs can convert one base to another: a C-G base pair becomes T-A, or A-T can be converted to G-C. This conversion happens without cutting the double-stranded DNA, without invoking DNA repair mechanisms that follow double-stranded breaks or using a donor template. The first generation of base editors from the Liu lab used a cytidine deaminase (APOBEC1) that was fused to catalytically impaired Cas9 (dCas9). Each next generation of base editors improved editing efficiency or specificity or both.

Addgene has helped labs iterate, optimize and reoptimize tools for editing genes ⁵. As of 23 December 2022, they had 430 CRISPR–Cas9 plasmids designed for base-editing in mammals, 30 for bacteria, 71 for plants, 30 for yeasts, and two for zebrafish.

Recently, it was announced that it was used to cure a form of leukemia (T-cell acute lymphoblastic leukemia, T-ALL) in a 13-year-old girl ⁶. She had already received all current conventional therapies for her cancer, including chemotherapy and a bone marrow transplant. Unfortunately, her disease came back and there were no further treatment options available as part of routine care. She was the first patient to be enrolled in a new clinical trial. She was admitted to the Bone Marrow Transplant (BMT) Unit at the Great Ormond Street Hospital for Children. She received genetically modified T-cells containing a chimeric antigen receptor (CAR) that originally came from a healthy donor. These cells had been edited using new base editing technology to allow them to find and kill the cancerous T-cells without attacking each other. Just 28 days later, she was in remission and went on to receive a second bone marrow transplant to restore her immune system. Now, six months after leaving the BMT, she is doing well at home and recovering with her family. She is continuing her post-BMT follow-up at GOSH. Base editing is an even more precise gene editing technique than CRISPR and has fewer risks of unwanted effects on the chromosomes and thus less risk of side effects. Researchers at Great Ormond Street and University College London were able to take healthy donor T-cells and remove two gene markers that prevent the modified cells from being destroyed by either the patient’s immune system or chemotherapy ⁶.

Scientists had already eliminated leukemia in several patients by converting their cytotoxic T cells into specific killers of cancer cells ^7,8. They used a lentiviral vector to express chimeric antigen receptors (CARs) in patients’ cytotoxic T cells. Extracellular and intracellular domains were fused into a new, synthetic receptor for the T cells. The gene for the CAR was delivered by a lentivirus vector, which was made by using the backbone of the HIV-1 virus that was no longer pathogenic and couldn’t cause AIDS. Because the natural host for HIV-1 is the T-lymphocyte, the lentivirus vector specifically targeted the cancer cells and multiplied in them. The patients received between 15 million and one billion cytotoxic T-lymphocytes (CTLs). These CTLs rapidly multiplied about 100-fold ⁸.

Engineered immune cell therapy is improving cancer therapeutics ⁹. The United States Food and Drug Administration (FDA) approval of two chimeric antigen receptor (CAR)-T cell products, axicabtagene ciloleucel (Yescarta ®) and tisagenlecleucel (Kymriah®), for the treatment of patients with B cell malignancies. However, several genetic edits will be needed to improve the efficacy of CAR-T cell therapies if they are to treat refractory malignancies successfully, especially solid tumors. Off-target effects of CRISPR–Cas9-mediated multiplex editing are affecting its safety and application in the clinic. Novel base editing technologies offer a promising and safer alternative for simultaneous editing that could enhance engineered immunotherapies for targeting solid tumors and other complex human diseases. Gene editing is a fast moving field, with new developments being published almost every week, which push the development of cell therapeutics forward ⁹.

Prime editing (PE) is a newer technique that uses a specialized enzyme called a prime editor to make precise changes to the DNA sequence of a gene ¹⁰. Like gene editing, prime editing can be used to correct genetic defects or to introduce new traits into an organism. However, prime editing has several advantages over traditional gene editing techniques. For example, prime editing can make changes to the DNA without the need for cutting the DNA, which reduces the risk of unintended changes to the genome. Prime editing can also make a wider range of changes to the genome than traditional gene editing techniques, including changes to multiple base pairs at once and the conversion of one base pair to another. A catalog of 348 causative variants regulating important agronomic traits in rice was analyzed. The results suggest that over 85% of them can be introduced precisely into the rice genome by prime editors. Moreover, prime editors produce far fewer off-target edits than base editors ¹⁰.