KEY INNOVATIONS IN GENE EDITING TECHNOLOGY_ITSAPPLICATIONS AND FUTURE

 

   Babies have been born who will not get AIDS. Corn which would resist drought is growing in fields. Our own immune cells which would go and trace your cancer cells and destroy them have been created. Mushrooms which will not brown quick would soon be found  in the shelves of super markets. Tomatoes which will not ripen soon too. Some tomatoes may be ‘hot’ like their cousin chilly pepper.  Deadly  genetic disease muscular dystrophy may be manipulated.  The genes causing Cystic fibrosis, another genetic disease could be altered. A cocktail could be straight injected to your tissues to cure a genetic disease. Gene editing gathered novelty in last few years and its implications have already changed the world. 

    An immunity trick bacteria play to ward off secondary infection by viruses has been adapted for editing genes of other organisms. At each infection event the bacteria would keep a little piece of viral DNA in their genome. When the same virus infects again this piece would remind the bacteria to avoid the infection and would resist it. The CRISPR-Cas9 system is a very specific enzyme action maneuver which could cut the DNA of the virus and would do the same in other cells.  Researchers create a small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence. CRISPR-Cas9 system has replaced other gene editing systems completely and has revolutionized pure and applied science alike. Previous techniques lacked specificity or generated off-target effects. Genes could be deleted out (called ‘knock out’) or replaced by new ones (‘knock in’) as needed. In a mere seven years, Cas9 has shown itself to be a formidable gene editor, employed in humans, plants, animals and bacteria to quickly and accurately cut and splice DNA, transforming biology and opening new avenues for treating disease.

    Cas9  is not the only enzyme used in this techniques. Other similar enzymes with varied functions are being found out. While Cas9 targets and cuts DNA, making permanent changes to the genome, Cas13 systems alter RNA, molecules that translate instructions from DNA to make proteins in the body and so it can correct  DNA mutation consequences by exchanging RNA. This is now being applied for finding treatment for muscular dystrophy and Parkinson’s disease. The approach is becoming more popular with scientists because it's reversible—RNA naturally degrades and RNA editing does not tamper with the genome. But it is effective in making the correct protein. While Cas12 would target double stranded DNA, Cas14 detects single stranded DNA. Another newly found member is CasX, a smaller brother in the series  and so would easily go into human cells and would not cause an immune reaction in humans. Many different kinds of diseases could be targeted by selecting appropriate enzyme.

CRISPR corrects genetic diseases-muscular dystrophy, cystic fibrosis and others 

      Genetic diseases cannot be cured. Treatments are inefficient since DNA mutations cannot be altered. CRISPR technology is disproving this norm. Genome editing has tremendous promise for therapeutic correction of genetic errors in human cells.  A mutation in dystrophin gene causes Duchenne muscular dystrophy (DMD) characterized by degeneration of cardiac and skeletal muscles, loss of ambulation and premature death. There is no effective long term therapy for DMD and the only drug allows for restoration of  less than 1% of the normal level of dystrophin protein. Now it has been found that using CRISPR method the mutation could be corrected in mice and human cells. The possibility arises that these ‘corrected’ cells could be implanted in the body. Thalassemia, another genetic disorder makes imperfect hemoglobin molecules which could cause an early death.  Frequent blood transfusions are required and the patients are challenged by immunological issues. Now a therapy regimen is at sight: induced pluripotent stem cells from the patient could be genetically corrected using CRISPR method and reinfused to the patient. Cystic fibrosis, another deadly genetic disease is caused by two types of mutations in the CFTR gene. Genome editing strategies have demonstrated the removal of these mutations and bringing up the gene to its correct function. This includes a novelty too: the editing has been done on patient derived organoids which would enable reimplation to the body, defying rejection by immune system. A single, point mutation in hemoglobin causes sickle cell anemia which drastically deforms red blood cells and results in mortality and morbidity worldwide.  It has been shown that gene editing strategies performed in blood forming stem cells could correct this mutation. A combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for effective treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. The successful disruption of CCR5 and CXCR4 genes (these genes are responsible for the production of proteins which the virus uses to bind to the cells) in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. 

Early diagnosis-beyond the gene editing function

  The Cas enzymes have been found to have a novel utility. They are not being used for chopping DNA and inserting the desired gene, but for detecting the presence of particular piece of DNA or RNA. This has been beneficially utilized to diagnose diseases. In an inexpensive way, that is the catch. CRISPR tool famous for its current function is being shifted to assume responsibility as a diagnostic tool.  The test relies on the system’s ability to hunt down genetic snippets, which could be RNA. Researchers at MIT have come with a new test method, aptly named SHERLOCK. While in the original CRISPR system Cas9 is used, here another variant enzyme Cas13 is employed. Cas13 cuts its target genetic sequence and then starts slicing up RNA indiscriminately. For editing genes this could be a problem but for diagnostics this is a big advantage: all those cuttings could serve as signals. In SHERLOCK, RNA molecules are used as signals when they are sliced by Cas13. The cut RNA triggers the formation of a dark band on a paper strip that indicates the presence of whatever genetic sequence CRISPR was engineered to find. This is as simple as using a pregnancy strip.

 

       The benefits of this test are varied. First of all it is cheap, half the prize of routinely used PCR tests, so affordable. The test is easy to execute and does not require elaborate equipment systems.The results are obtained immediately. The diagnosis could be done way before the symptoms are evident, so many could be saved from the disease and also help curb further transmission. In Nigeria attempts are underway to use the test kit for Lassa virus infection. Trial tests are being done to diagnose dengue virus, Zika virus and cancer associated  strains of human papilloma virus (HPV). Early diagnosis of HPV would help to curb the rising death toll from cervical cancer in African countries. A study is being done in Congo to extend the test for Ebola virus detection.   

CRISPR knockout, CRISPR interference, CRISPR activation- Novelty in drug discovery 

     Assessments of new compounds for drug development are notoriously long and costly; they typically span more than a decade and exceed a billion dollars. Even then only a small percentage would reach the market. The challenge is to identify the disease target and testing therapeutic efficacy. CRISPR technology has changed this scenario. Now we can activate a gene, knock it out or interfere with its function efficiently and check what changes this would impart to the cell. Does it mimic a disease? Does it curb the symptoms? What chemical or drug would effectively silence the gene? Thus the whole process is leading  to novel drug discovery patterns. Perturbations that exacerbate or hinder a disease can reveal potential drug targets. 

           Knock out or knock in of genes in cultured cells can be used to ensure that the target is limited to the disease of interest. If the target is druggable then a screening campaign is initiated to search for potential therapeutic candidates. Many compounds could be used and  screened in a massive scale.  Gene editing has improved cell-based screens by enabling scientists to more accurately generate cell lines with mutations relevant to the disease of interest.  Initial screens can accurately eliminate ineffective compounds. Hits identified through the initial screens are validated through a variety of cell-based assays. Stem cells, primary cells and organoids with appropriate genetic backgrounds could be used to recreate genetic variation associated with a disease.  The validation of hits would narrow the candidate drugs to a smaller range. These drugs could be optimized and tested for safety. CRISPR has accelerated process in this phase by enabling the generation of cellular and animal models with multiple mutations. Thus the cause of a disease could be identified by the initial gene editing experiments on cell lines, drugs have been screened and optimized, animal models with that specific gene abnormality has been created and drugs already designed. Clinical trials and government approval are the  remaining procedures. It is evident that CRISPR has enhanced the speed and precision of screening, helping scientists uncover new drug targets and better understand the genome. 

Target validation in cancer cells-drug resistance in chemotherapy reversed 

        A library could be made of an array of single stranded guide RNA and this could be used to transform or transduct cells which already have Cas9 enzymes active in them. That means whichever cell will receive a specific guide RNA, the corresponding gene would be deleted by this enzyme, as one of the example. When these cells are grown in multi-well pates clones of cells with different gene knock outs (or knock ins) could be produced. These cells are then used for screening drugs, depending on whether the drug kills a specific clone. Now the gene responsible for that drug effect could be identified. Modifications and elaboration of this technique are widely being used in deciphering exact gene target in cancer chemotherapy. New drugs which target genes found to be involved in carcinogenesis is revolutionizing cancer therapy. 

   Gene editing tools have been found to reverse cancer resistance to chemotherapy. Cancer causing genes have a tendency to mutate and the genes altered in this way will not respond to chemotherapy. This is an example of survival mechanism tumor cells adopt. A high rate of chemotherapy resistance has been observed in lung cancer tumors. More than two-thirds of lung cancers are resistant to the drug carboplatin, for example. While most of the CRISPR research is heavily focused on treating genetic diseases by altering genes, here attempts are being made to change the genes of the tumor, not the person. For example the gene NRF2 which controls many other genes which allow cancer cells to develop resistance to chemotherapy could be turned off using CRISPR method.  Thus the resistance is muted and all the tumor cells would respond to even low levels of carboplastin. When these genetically altered tumor cells were implanted to lung tumor in mice, the tumor growth was retarded.  This approach has been heralded as a very promising one since CRISPR allows manipulation of genes with more accuracy and precision.  This could be extended human tumor treatment and raises only fewer ethical concerns, since the method avoids manipulating human genes.

Let your own immune cells kill your cancer cells

         Cancer cells can hide cleverly from being noticed by our immune cells which would attack them otherwise. Manipulating the immune cells, especially one type of them called T cells would effectively annihilate cancer cells if given a chance. These T cells are destructive by nature and they have special proteins on their surface to recognize our own cells and leave them alone. But cancer cells adorn many of the proteins on them declaring themselves as our own so that T cells will not attack them. The system is called check point inhibitors. There are two proteins on the surface of T cells PD-1 and CTLA-4 which would help them recognize our own cells and cancer cells would trick the T cells by adorning receptors for these proteins. The strategy now is to block these two proteins on the T cell surface so that they can attack cancer cells. Gene editing methods have been devised to block the production of these two proteins. Using CRISPR technology, PD-1 knockout engineered T cells have been effectively used to treat non-small cell lung cancer. Trials are being conducted in prostate, renal and bladder cell cancers and esophageal cancer.

         Generating chimeric antigen receptor (CAR) T cells by CRISPR is an ex vivo (outside body) approach in clinical trials. In this method T cells from the patient’s blood are collected and then genetically altered by inserting genes which would direct synthesis of special proteins to be beset on T cell surface. These cells are expanded in culture and injectec back into the patients’s blood. These proteins (antigens) are specifically designed (that is why called ‘chimeric’)  to attack and kill the cancer cells. Trials are underway to effectively treat multiple myeloma, melanoma, synovial sarcoma and myeloid cell liposarcoma.

Super babies-CRISPR Frankensteins?

Genetically altered human beings have become a reality. He Jiankui, a Chinese scientist manipulated human embryos to get a specific protein CCR5 removed from the surface of immune cells. This protein is needed for AIDS virus to get attached to these cells. Thus babies –twins named Lulu and Nana-  were born who will not get infected with human immunodeficiency virus (HIV) which means they are naturally immune to that virus. Their father carries HIV and now spreading the virus to generations to come has been prevented. The prospect of this irrevocable genetic change is of concern. Since the advent of CRISPR as a genome editor 5 years earlier, the editing of human embryos, eggs or sperm has been hotly debated. The core issue is whether such germline editing would cross an ethical redline because it could ultimately alter our species. He Jiankui’s creation was phenomenal but violated the international prohibition rule of altering human embryos. This let people all over the world confront an uncomfortable truth: that regulations and scientific community’s efforts to control CRISPR’s powers had failed.

                 This was a clear example of technology defying laws and ethics. A revelation that if a technique is available it would be misused. A particular group, society or country could make ‘tailor made humans’ to satisfy the whims and fancies of them. The possibilities of altering populations could be devastating. Skin color could be changed, sports performance could be increased, sexual desires altered, aggressiveness or timidness enhanced and stories of science fiction movies could be a reality soon. The prime issue is how to regulate human embryonic gene editing. Manipulation of human embryos by gene editing is permitted in many countries but implanting them on fertile females is prohibited.  Who should or who can control gene editing? What are the needs behind human gene editing? What all genes could be edited? The questions are many and relevant.

Sleepless monkeys, muscular dogs

        Through gene editing scientists in China have already produced monkeys which have sleep problems, as a prelude to study sleep disorder. China’s researchers have produced many other animal varieties-dogs, rats, pigs and rabbits-with altered or deleted genes and the promise is to bring up high quality meats, disease –resistant livestock and new medical treatments and organs for human transplantation. Gene edited pigs would have organs ready for transplantation to humans: the organs will not be rejected. The gene which makes a particular protein which would initiate a rejection reaction in humans has been deleted in these pigs. These animals have been engineered to  age prematurely or develop neurodegenerative afflictions that mimic diseases such as Alzheimer’s, Parkinson’s Lou Gehring’s disease and Huntington. In some animals CRISPR technology has been used to knock in a humanized gene for albumin, a blood product given in case of traumatic shock or liver failure. Pigs have been engineered to have 5% less white fat which makes for leaner meat. A gene added with CRISPR made pigs resistant to classical swine fever. While some are altered to resist the costliest infection, porcine reproductive and respiratory syndrome virus. Dogs which can jump higher or run faster have been created proving that canines also yield to gene editing manipulations.

 

CRISPR repairs your body parts

       If the technique works in cultured cells why can’t the system do the same job  inside your body? Yes, that could happen. When the cocktail containing the appropriate guide RNA and the Cas9 enzyme system ingredients is injected into our cells the gene editing will perform perfectly. This possibility has been effectively utilized in treating retinis pigmentosa and LCAA10 (Leber congenital amaurosis10). Retinis pigmentosa is caused by a mutation in one of the proteins needed for vision -rhodopsin. A single subretinal injection of guide RNA/Cas9 plasmid generated  specific disruption of this mutated gene. Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene and now this faulty gene could be edited out by injection of CRISPR/Cas9 system into retina. 

CRISPR’d crops

 With shrinking agricultural lands and increasing population, food production and  resources need to be revolutionized. Gene editing provides an effective method to advance production and has great potential. Researchers are pursuing more ambitious changes: wheat with triple the usual fiber, or that's low in gluten. Mushrooms that don't brown, and better tasting tomatoes. Drought-tolerant corn and rice that no longer absorb soil pollution as it grows. Dairy cows that don't need to undergo painful de-horning, and pigs immune to a dangerous virus that can sweep through herds. A larger number of small companies can afford to get into the sector because products generated by gene-editing techniques such as CRISPR have not been subjected to rigorous rules of conventional GMO plants.

Scientists hope that gene editing eventually could save species from being wiped out by devastating diseases like citrus greening, a so far unstoppable infection that's destroying Florida's famed oranges.

       The following are some of the genetic manipulating  attempts which would change crop yield, quality, pest resistance and overall production using gene editing technology:

·         Canola and soybean with increased oil content. In yellow-flowered oilseed Camelina sativa three genes are being inactivated to increase oil content and field tests are underway.

·         Fruits and vegetables would soon be more eater friendly. Seedless watermelons and naturally baby-cut carrots are examples.

·         Tomatoes which may not taste fresh after shipping long distances would be history.  Gene edited tomatoes are being developed which would provide high yield, ship long distance  and taste like it came from your garden.

• Waxy corn, a variety edited to contain elevated levels of amylopectin and reduced levels of amylase would be on the market soon. High amylopectin cornstarch would improve freeze-thaw properties of frozen foods and make canned foods and dairy products more creamier. This novel cornstarch could be a better adhesive to stick labels on bottles. 

• Naturally decaffeinated coffee may appear in stores soon. 

• Wheat, soybean and corn which are disease resistant. Pesticide use will be drastically reduced.
• Mushrooms and potatoes which are slow to brown are already present.
• Disease resistant bananas would be soon a reality. 

• Row crops edited for high yield, stress resistance and herbicide tolerance are ready for filed tests. 
• Inhibition of a specific protein production which makes wheat susceptible to powdery mildew  makes that wheat variety resistant to the disease. 

• A healthier soybean oil has been created by CRISPR technology. It has zero trans fats, 80% oleic acid, three times the fry life and extended shelf life. 

   Now that new varieties of Cas enzymes like Cas12 and Cas13 are being introduced,  regulating  multiple genes at multiple genomic sites is possible and so in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology. Law makers and scientists are debating whether these crop products should be labeled ‘gene edited’. 

A CRISPR bright horizon 

     CRISPR system has not only revolutionized genome engineering but has also brought the possibility of translating these concepts into a clinically meaningful reality.

Tissues for transplantation are getting ready in CRISPR labs. Engineered pig corneas or insulin making pancreatic islet cells could be infused into humans. By the end of next year researchers may test a pig kidney with nine modified genes in people on dialysis. 

  Cancer drugs have more specificity and new target genes are being discovered.          Since many of the genes causing cancer have been specified these genes could be replaced by death promoting genes. Methods are being developed to avoid off target events such as avoiding normal genes being affected. Another promising application of the CRISPR system is personalized therapy which requires rapid and systematic screening to identify the genotype specific changes in the patient’s genome. CRISPR genome editing is spreading its impact in the clinics in unforeseen ways. For example plastic and reconstructive surgery regimens are adapting gene editing techniques to manage wound healing, craniofacial malformations and tissue engineering. CRISPR technique may help to correct mutations and prevent the development of cleft palate, cleft lip and other congenital malformations. 

 CRISPR is revealing more secrets hiding in the dark corridors of cancer therapy. Recently it has been found that some cancer drugs hit unexpected targets not intended initially. This brings in a caveat that the interpretations of drug effects need to be scrutinized thoroughly. 

   But ever since the Chinese CRISPR babies incidence scientists, law makers and the public have grave concerns about the priorities and usage of the technique.  Some are calling for a global moratorium on all research that would tinker with the genes of human embryos. The future of embryonic editing would depend on the need for it. Since the potential of the technique is expanding in logarithmic scale with new innovations, the appeal is tantalizing. Who would not desire their children to be free of a genetic disease?