We are all protein robots. Every last being on Earth is nothing more than a creature built from amino acids, with a single purpose to reproduce and pass on the code that built us and decides what we do every day. For centuries, that code remained inviolable and mysterious, some creation of a being beyond our greatest minds. That code has been cracked by the Human Genome Project (HGP) along with the private company Celera Genomics, and now, with the advent of CRISPR/Cas9 technology, genetic engineering is cheaper and more accessible than ever before. So, how does genetic engineering work, what can it achieve, and what are some of the possible pitfalls of this new technology?
The history of genetic engineering goes back way further than most of us may think. We may think that it began with the discovery of the double helix, or perhaps with the first successfully synthesized DNA in vitro, but in reality, we have been modifying the plants and animals that live around for thousands of years. Artificial selection, or selective breeding, is the process in which humans breed certain animals or plants with certain traits to get a desired specimen. Wolves to dogs, aurochs to cows, and every single crop we consume in modern times, are all products of artificial selection.
Modern genetic engineering began with the discovery of the double helix by James Watson and Francis Crick in 1953, defining genetics to the modern day. A lesser-known individual, who was crucial to the discovery of the double helix but is often forgotten, is Rosalind Franklin. Without her x-ray diffraction images of DNA proteins in the early 1950s, James and Francis would’ve never have considered the idea of a double helix structure for DNA.
In 1962, Osamu Shimomura isolated the GFP (Green Fluorescent Protein), and researchers Martin Chalfie and Roger Tsien developed it into one of the most important tools for genetic research and engineering. GFP allowed scientists to determine which cells expressed the target gene they were researching, and allowed scientists to monitor the inside of living cells without harming them.
The first experiments in gene splicing came after the discovery of DNA ligases, which are crucial for the repair and replication of genetic material in all organisms, which by catalyzing the formation of a phosphodiester bond (The covalent bond that links the sugar and phosphate groups in nucleic acids), scientists can join DNA strands together, leading to the creation of recombinant DNA in the early 70s. The discovery of restriction enzymes and modification enzymes by Werner Arber, who observed that bacterium were capable of fighting off phage infection by cutting out the foreign DNA, hypothesized the existence of a “restriction” enzyme, which identified and removed foreign DNA, and the “modification” enzyme, which protects host DNA from removal. His discoveries paved the way for the first chimeric recombinant DNA in 1972.
Genetic engineering involves a wide variety of constantly changing approaches to modifying the DNA sequence in genomes. Homologous recombination can be used to target certain sequences in mouse embryonic stem cell genomes or other cultured cells, but this is slow, inefficient, and reliant on drug positive/negative selection in cell culture. Other commonly used methods include microinjection, transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors to produce transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but it has many drawbacks despite its efficiency. Currently, the most effective method is based upon guided endonucleases, because these are capable of targeting specific DNA sequences. Now with the onset of CRISPR/Cas9 or clustered regularly interspaced short palindromic repeats technology, endonuclease-mediated gene targeting has become the most widely used method to engineer genomes, superseding the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases.
CRISPER/Cas9 is the most effective, efficient, and accurate method of genome editing in living cells, revolutionizing genetic engineering from a slow, tedious, and expensive, hit-or-miss process into a precise scientific measurable scientific tool, so how exactly does this miraculous system work?
The CRISPR/Cas systems can be divided into six classes based on the structure and function of the Cas-proteins. Class I consists of types I, III, and IV, while Class II contains types II, V, and VI. Class I systems consist of multi-subunit Cas-protein complexes, while class II systems utilize a single Cas-protein. The structure of type II CRISPR/Cas9 is relatively simple, and so it has been thoroughly studied and extensively used in genetic engineering. The Cas9 protein was the first Cas protein used in genome editing, and it was extracted from Streptococcus pyogenes (SpCas9).
SpCas9 is a large multi-domain DNA endonuclease responsible for cleaving the target DNA to form a double-stranded break and is called a genetic scissor. Cas9 consists of two regions called the recognition (REC) lobe and the nuclease (NUC) lobe. The REC lobe consists of REC1 and REC2 domains responsible for the binding guide RNA, whereas the NUC lobe is composed of RuvC, HNH, and PAM interacting domains. RuvC and HNH are used to cut each single-stranded DNA, while PAM interacting domain confers RAM specificity and is responsible for initiating binding to target DNA.
Guide RNA is made up of two parts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). CrRNA is an 18-20 base pair in length that specifies the target DNA by pairing with the target sequence, whereas tracrRNA is a long stretch of loops that serve as a binding scaffold for the Cas9 nuclease. In prokaryotes, guide RNA is used to target viral DNA, but in CRISPR, it can be synthetically designed by combining crRNA and tracrRNA to form a single guide RNA (sgRNA) to target almost any gene sequence supposed to be edited.
Guide RNA (gRNA) and Cas9 proteins are the two essential parts in CRISPR/Cas9. CRISPR/Cas9 genome editing contains three steps: recognition, cleavage, and repair. The sgRNA (Single Guide RNA) recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas9 nuclease makes double-stranded breaks at a site 3 base pairs upstream to a Protospacer Adjacent Motif (PAM), then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms.
CRISPR/Cas9 was invented in 2012, yet in just a few years, it has already been explored for a wide number of applications and has had a massive impact in medicine, agriculture, and biotechnology.
There are more than 6000 known genetic disorders, yet many of these diseases lack effective treatments. Gene therapy is the process of replacing the defective gene with outside DNA and editing the mutated gene at its native location. It is the latest development in the revolution of medical biotechnology. From 1998 to 2026, more than 50 gene therapies, including CRISPR/Cas9, have been approved for the treatment of human diseases.
Ever since its discovery, CRISPR/Cas9 gene editing has held the promise of curing most of the known genetic diseases such as; sickle cell disease (SCD), β-thalassemia, cystic fibrosis, and muscular dystrophy. CRISPR/Cas9 treatment for SCD and β-thalassemia have also been applied in clinical trials.
SCD is an autosomal recessive genetic disease of red blood cells, which occurs due to point mutation in the β-globin chain of hemoglobin, which leads to sickle hemoglobin (HbS). During the deoxygenation process, HbS polymerization leads to severe clinical complications like hemolytic anemia.
The two main approaches that CRISPR/Cas9 is used to treat SCD is either through direct repair of the hemoglobin S gene or by boosting fetal y-globin. However, the most common method used in a clinical trial is based on the approach of boosting fetal hemoglobin. First, bone marrow cells are removed from patients and the gene that turns off fetal hemoglobin production called B-cell Lymphoma 11A (BCL11A) is disabled with CRISPR/Cas9. Then the gene-edited cells are infused back into the body. BCL11A is a 200 base pair gene found on chromosome 2, and its product is responsible for switching y-globin into β-globin chain by repressing y-globin gene expression. Once this gene is disabled using CRISPR/Cas9, the production of fetal hemoglobin containing y-globin in the red blood cells will increase, alleviating the severity and manifestations of SCD.
The first CRISPR-based therapy in the human trial was conducted to treat patients with refractory lung cancer using modified T-cells. Researchers first extract T-cells from three patients’ blood, then they engineered them in the lab using CRISPR/Cas9 to delete genes (TRAC, TRBC, and PD-1) that interfered with the T-cells’ ability to fight cancer cells. Then the modified T-cells were infused back into the patients. The patients exhibited no side effects from this treatment, and the engineered T-cells could be detected up to 9 months post-infusion.
Another focus area for CRISPR/Cas9 research is the treatment of HIV. In May 2017, a team of researchers from Temple University demonstrated that HIV-1 replication can be completely shut down and eliminated from infected cells through the removal of the HIV-genome. CRISPR/Cas9 technology can also be used to block HIV entry into host cells by editing chemokine co-receptor type-5 (CCR5) genes in the host cells. Recent in vitro trials conducted in China report that genome editing of CCR5 by CRISPR/Cas9 showed no evidence of infection on cells, concluding that edited cells could effectively be protected from HIV infection.
CRISPR/Cas9 also plays an important role in agriculture, improved nutritional value, greater shelf life, resistance to natural disasters and disease, CRISPR/Cas9 has been used to do that and more. CRISPR/Cas9 may even be the solution to the world’s food crisis with its ability to restore food supplies, help plants survive harsh conditions, and improve overall health of the plants.
Despite CRISPR/Cas9’s great promise and potential as a genome-editing system, it has been hampered by several challenges that must be addressed during the process of application. Immunogenicity, a lack of a safe and efficient delivery system to the target, off-target effects, and ethical issues have all been major barriers to further use of CRISPR/Cas9 in clinical applications.
The biggest issue is the host’s immune response. Since the components of the CRISPR/Cas9 system are derived from bacteria, the host immune system can elicit a response against these components. Researchers found that there were both pre-existing humoral (anti-Cas9 antibodies) and cellular (anti Cas9 T cells) immune responses to Cas9 proteins in healthy humans. Therefore, the detection and reduction of the immune response against Cas9 is still one of the most important challenges in clinical trials.
Another major issue is the safe and effective delivery of the components into the cell. There are currently only three methods of delivering the CRISPR/Cas9 complex into cells: physical, chemical, and viral vectors. Non-viral methods are more suitable for ex-vivo CRISPR/Cas9 based gene editing therapy.
The physical methods of delivering CRISPR/Cas9 can include; electroporation, microinjection, hydrodynamic injection, and more. A major downside to electroporation and microinjection is the damage they cause to cells, and while hydrodynamic injection has shown promise on animals, being simple, fast, efficient, and versatile, it has not yet been used in clinical applications due to possible risks.
Chemical methods of CRISPR/Cas9 delivery involve lipid and polymer-based nanoparticles. Lipid nanoparticles/liposomes are spherical structures composed of lipid bilayer membranes and are synthesized in aqueous solutions using Lipofectamine-based reagents. The positively charged liposomes enveloped by the negatively charged nucleic acids facilitate the fusion of CRISPR/Cas9 across the cell membrane into cells. Polymeric nanoparticles are the most widely used carriers of CRISPR/Cas9 components. Like lipid nanoparticles, polymer-based nanoparticles can also transverse the complex in the membrane through endocytosis.
Viral vectors are the natural experts for in vivo CRISPR/Cas9 delivery. Many viral vectors such as; adenoviral vectors (AVs), adeno-associated viruses (AAVs), and lentivirus vectors (LVs) are currently being widely used as delivery methods due to their higher delivery efficiency relative to physical and chemical delivery methods. However, the limited virus cloning capacity and the large size of the Cas9 protein remains a problem.
In conclusion, genetic engineering has evolved from simple selective breeding into one of the most powerful scientific technologies ever developed. With CRISPR/Cas9, scientists can now edit genes with unparalleled precision, opening the door to curing genetic diseases, improving agriculture, and advancing biotechnology in previously impossible ways. However, there are many serious scientific, medical, and ethical challenges. As this technology continues to grow, society must carefully balance innovation with responsibility to ensure the safe and responsible use of genetic engineering.
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