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1) the first benefit to this gene modification is that it can help improve the ability of immune cells to attack cancer

2) the second benefit can help by helping treat diseases like Alzheimers.

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1-The process to make dogs stronger can help prevent human diseases.
2- Helps prevent muscular dystrophy and Parkinson's diseases.

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1) It can cause mutations in other genomes.

2) The unavailability of PAM (protospacer adjacent motif, is a short specific sequence following the target DNA sequence) in the desired gene loci.

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1.  Other applications include the production of nonprotein (bioplastic) or nonindustrial (ornamental plant) products.
2. increased crop yields, reduced costs for food or drug production

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1) The CRISPR gene editing system can solve problems of treating cancer in a more efficient way. It removes proteins for genes!

2) The CRISPR gene editing system also can easily cause mutations which is the main problem. Scientists are working to find a solution to this.

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1- The process can cause a lot of pain to the dog while testing
2- can result in painful births

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https://www.cnn.com/2015/10/28/world/china-mutant-dogs-genetic-engineering/index.html

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genetically engineered dogs in china - cnn

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Super-strong, genetically engineered dogs in China - CNN

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Scientists in China have created super-strong dogs through genetic engineering. Could this lead to cures for Parkinson's disease and muscular dystrophy?

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Personally, I think its a bad idea to try to go in and modify our own genes. Even though it could help get rid of cancers like lung cancer as tested, the side affects which could be things like mutations. It can also help get rid of diseases like Alzheimers but if im old to the point of having said Alzheimers id rather keep those instead of trying to modify my genes to get rid of it.

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https://sitn.hms.harvard.edu/flash/2015/from-corgis-to-corn-a-brief-look-at-the-long-history-of-gmo-technology/

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Summary: To date, scientists have engineered bacteria that produce medication-grade drugs, crops with built-in pesticides, and beagles that glow in the dark. While these are all relatively recent advances in scientific technology, humans have been altering the genetics of organisms for over 30,000 years. How did the original practice of selective breeding evolve into the concept of genetically modified organisms, as we know it today? Innovators, motivated by some of the world's most critical problems, have paved the way for GMOs - a path that leads to an unimaginable array of benefits, but also raises extremely important questions.

The concept of "genetically modified organisms," or GMOs, has received a large amount of attention in recent years. Indeed, the relative number of Google searches for "GMO" has more than tripled since late 2012 [1]. However, humans have been genetically modifying organisms for over 30,000 years [2]! Clearly, our ancestors had no scientific laboratories capable of directly manipulating DNA that long ago, so how did they do it, and how have GMOs become such a popular topic?

Ancient Genetic Modification

While our ancestors had no concept of genetics, they were still able to influence the DNA of other organisms by a process called "selective breeding" or "artificial selection." These terms, coined by Charles Darwin, describe the process of choosing the organisms with the most desired traits and mating them with the intention of combining and propagating these traits through their offspring. Repeated use of this practice over many generations can result in dramatic genetic changes to a species. While artificial selection is not what we typically consider GMO technology today, it is still the precursor to the modern processes and the earliest example of our species influencing genetics.

The dog is thought to be the first organism our ancestors artificially selected. Around 32,000 years ago, while our ancestors were still hunters and gatherers, wild wolves in East Asia joined groups of humans as scavengers. They were domesticated and then artificially selected to increase docility, leading to dogs that are closely related to what are currently known as Chinese native dogs [2]. Over millennia, various traits such as size, hair length, color and body shape were artificially selected for, altering the genetics of these domesticated descendants of wolves so much that we now have breeds such as Chihuahuas and corgis that barely resemble wolves at all! Since this time, artificial selection has been applied to many different species and has helped us develop all sorts of animals from prize-winning racehorses to muscular beef cattle.

Artificial selection has also been utilized with a variety of plants. The earliest evidence of artificial selection of plants dates back to 7800 BCE in archaeological sites found in southwest Asia, where scientists have found domestic varieties of wheat [3]. However, one of the most dramatic and prevalent alterations in plant genetics has occurred through artificial selection of corn. Corn, or maize, began as a wild grass called teosinte that had tiny ears with very few kernels [4]. Over the hundreds of years, teosinte was selectively bred to have larger and larger ears with more and more kernels, resulting in what we now know as corn. A similar process has given us large heads of broccoli, bananas with nearly unnoticeable seeds, and apples that are sweet and juicy.

Although artificial selection is an ancient process that is still used today, most current conversations regarding GMOs refer to a much more modern process of altering the genetics of organisms.

The Birth of Modern Genetic Modification

An enormous breakthrough in GMO technology came in 1973, when Herbert Boyer and Stanley Cohen worked together to engineer the first successful genetically engineered (GE) organism [5]. The two scientists developed a method to very specifically cut out a gene from one organism and paste it into another. Using this method, they transferred a gene that encodes antibiotic resistance from one strain of bacteria into another, bestowing antibiotic resistance upon the recipient. One year later, Rudolf Jaenisch and Beatrice Mintz utilized a similar procedure in animals, introducing foreign DNA into mouse embryos [6].

Although this new technology opened up countless avenues of research possibilities, immediately after its development, the media, government officials, and scientists began to worry about the potential ramifications on human health and Earth's ecosystems [7]. By the middle of 1974, a moratorium on GE projects was universally observed, allowing time for experts to come together and consider the next steps during what has come to be known as the Asilomar Conference of 1975 [8]. At the conference, scientists, lawyers, and government officials debated the safety of GE experiments for three days. The attendees eventually concluded that the GE projects should be allowed to continue with certain guidelines in place [9]. For instance, the conference defined safety and containment regulations to mitigate the risks of each experiment. Additionally, they charged the principal investigator of each lab with ensuring adequate safety for their researchers, as well as with educating the scientific community about important developments. Finally, the established guidelines were expected to be fluid, influenced by further knowledge as the scientific community advanced.

Due to the unprecedented transparency and cooperation at the Asilomar Conference, government bodies around the world supported the move to continue with GE research, thus launching a new era of modern genetic modification.

Use of Genetically Engineered Organisms

In 1980, the U.S. Supreme Court of the ruled that scientists from General Electric could patent bacteria that were genetically engineered to break down crude oil to help with oil spill mitigation [10]. This ruling legally permitted ownership rights over GMOs, giving large companies the incentive to rapidly develop GMO tools that could both be useful and profitable.

Two years later, in 1982, the United States Food and Drug Administration approved the first human medication produced by a genetically modified organism. Bacteria had been genetically engineered to synthesize human insulin, allowing them to produce enough of the hormone to purify, package, and prescribe it to diabetes patients as the drug Humulin [11].

While uses for genetic engineering range from oil spills to medication, perhaps the most controversial application is for food production. The first field experiments of food crops that had been genetically modified using recombinant DNA technology began in 1987. After five years of extensive health and environmental testing, Calgene's Flavr Savr tomato became the first food crop to be approved for commercial production by the U.S. Department of Agriculture. These tomatoes were modified to include a DNA sequence that inhibited production of a natural tomato protein, increasing the firmness and extending the shelf life of the Flavr Savr variety.

In addition to making food more aesthetically pleasing, scientists have developed crops that are easier to for farmers to cultivate. In 1995 the first pesticide-producing crop was approved by the U.S. Environmental Protection Agency after rigorous testing [12]. A year later, Bt corn was approved, and now the majority of corn in the U.S. has the Bt toxin gene (see this article). Additionally, crops have also been genetically engineered to resist herbicides, making it easier for farmers to control unwanted plants in their fields. Perhaps the most famous herbicide resistant crops are the Roundup Ready or glyphosate-resistant plants (see this article). The first of these glyphosate-resistant crops was a variety of soybean, engineered by Monsanto in 1996. Now glyphosate-resistant technology has been applied to many other crops, including corn and sugar beets.

Scientists have also genetically engineered crops to increase nutrition value. For instance, Golden Rice was developed in 2000 with the goal to combat vitamin A deficiency, which is estimated to kill over 500,000 people every year (see this article)[13].

Although many species of animals have been genetically engineered, the vast majority of this technology is used for research purposes, and to date, there have been no GE animals approved by the FDA for use in food production [14]. However, in 2009, the U.S. FDA approved the first biological product produced by a GE animal, ATryn, a drug used to treat a rare blood clotting disorder [15].

Genetically Engineered Food Controversies

There have been many controversies regarding GE technology, with the majority relating to GE food. While some critics object to the use of this technology based on religious or philosophical bases, most critics object on the basis of environmental or health concerns. For instance, a 1999 publication showed Bt toxin had negative effects on butterfly populations in laboratory tests, leading to strong objections of Bt use, but follow-up studies in actual farming fields confirmed the safety of this technology [16]. In a different example, the economic stress of the poor yield of GE cotton crops in India over the late 1990s and early 2000s was associated by many organizations with a presumed increase in farmer suicides [17]. However, it was later concluded that suicide rates were actually unchanged after introduction of GE cotton, and that there were economic benefits of GE cotton for most Indian farmers [18].

During the same time frame, public awareness of the existence of GE foods increased, and calls for regulation of GE food grew louder, resulting in labeling requirements for GE food in many countries. Today, 64 countries have mandatory labeling laws for GE food [19]. However, the United States still does not have a mandatory, nationwide labeling law, although many advocacy groups are lobbying to enact one. These groups argue that labeling GE food is important for consumer choice and for monitoring unforeseen problems associated with the technology [20]. In contrast, groups opposing labels claim a law would unnecessarily eliminate consumer demand for current GE crops, causing steep increases in food price and resource utilization [20].

Although the debate about GE food is active, and there is no shortage of opponents to the technology, the scientific community has largely come together and concluded consumption of GE food is no more dangerous and eating traditionally selected crops [21]. This conclusion has not stopped businesses from capitalizing on the current fear of GE food. In 2013, Chipotle became the first restaurant chain to label menu items as "GMO," and in April of this year, the company announced the elimination of all ingredients made with GMOs, citing their "food with integrity journey" [22]. With cases such as this, it is safe to say the debate on GE food will continue for some time.

The Future of GMO Technology

There are countless potential uses of GE technology in development. These include plants with superior disease and drought resistance, animals with enhanced growth properties, and strategies for more efficient pharmaceutical production [23]. Likewise, GE technology itself is quickly advancing. Recently, researchers have developed a new technology called CRISPR, which takes advantage of bacterial systems to simplify genetic editing, allowing for easier development of GE organisms [24]. This technology could be used to expedite development of useful GE crops, facilitate disease elimination, or even alter entire ecosystems. Interestingly, recent advances in plant breeding techniques may increase the utility and rebound the popularity of the more traditional GMO method of selective breeding. Indeed, new drought resistant strains of various crops have been recently developed using traditional breeding methods [25].

The United Nations predicts that by 2050, humans will need to produce 70% more food than we currently do in order to adequately feed the global population (see this article) [26]. Indeed, innovative approaches will be required to solve this problem, and genetically engineering our food is a potentially useful tool. As scientists look forward at ways to create better crop survival, yield, and nutrition, it is important that we remember where all of this work began, and give credit to the pioneers who have made our advancements possible. Our ancestors that selectively bred wolves to eventually develop Corgis could not foresee that today we would be able to genetically engineer corn to withstand pests, herbicides, and drought. What is the future of GMO technology that we ourselves can't foresee now?
Gabriel Rangel is a Ph.D. candidate in the Biological Sciences in Public Health Program at Harvard University.
This article is part of the August 2015 Special Edition, Genetically Modified Organisms and Our Food.

References

1. "GMO Search Term." Google Trends, July 2015. https://www.google.com/trends/explore#q=GMO

2. Zimmer, C. "From Fearsome Predator to Man's Best Friend." New York Times, May 2013. http://www.nytimes.com/2013/05/16/science/dogs-from-fearsome-predator-to-mans-best-friend.html

3. Balter, M. " Farming Was So Nice, It Was Invented at Least Twice." Science, July 2013. http://news.sciencemag.org/archaeology/2013/07/farming-was-so-nice-it-was-invented-least-twice

4. "The Evolution of Corn." Genetics Learning Center, University of Utah, July 2015. http://learn.genetics.utah.edu/content/selection/corn/

5. Cohen, S. et. al. "Construction of Biologically Functional Bacterial Plasmids In Vitro." PNAS, November 1973. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC427208/

6. Jaenisch, R. and Mintz, B. "Simian Virus 40 DNA Sequences in DNA of Healthy Adult Mice Derived from Preimplantation Blastocysts Injected with Viral DNA." PNAS, April 1974. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC388203/
7. Committee on Recombinant DNA Molecules. "Potential Biohazards of Recombinant DNA Molecules."PNAS, July 1974. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC388511/?page=1

7. Berg, P. "Asilomar and Recombinant DNA." Nobel Media AB, August 2004. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1980/berg-article.html

8. Berg, P. et. al. "Summary Statement of the Asilomar Conference on Recombinant DNA Molecules." PNAS, June 1975. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC432675/pdf/pnas00049-0007.pdf

9. "Biotechnology." Encyclopedia Britannica, 2015. http://www.britannica.com/technology/biotechnology#ref926019

10. Altman, L. "A New Insulin Given Approval for Use in the U.S." The New York Times, October 1982. http://www.nytimes.com/1982/10/30/us/a-new-insulin-given-approval-for-use-in-us.html

11. "EPA's Regulation of Bacillus thuringiensis (Bt) Crops." U.S. Environmental Protection Agency, Februray 2014. http://www.epa.gov/pesticides/biopesticides/pips/regofbtcrops.htm

12. Ye et. al. "Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm." Science, January 2000. http://www.sciencemag.org/content/287/5451/303

13. "Genetically Engineered Animals: Consumer Q&A." U.S. Food and Drug Administration, June 2015. http://www.fda.gov/animalveterinary/developmentapprovalprocess/geneticengineering/geneticallyengineeredanimals/ucm113672.htm

14. "FDA Approves Orphan Drug ATryn to Treat Rare Clotting Disorder." U.S. Food and Drug Administration, February 2009. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm109074.htm

15. Sears, M. et. al. "Impact of Bt corn pollen on monarch butterfly populations: A risk assessment." PNAS, August 2001. http://www.pnas.org/content/98/21/11937.long

16. Heeter, C. "Seeds of Suicide: India's Desperate Farmers." Frontline World: PBS, July 2005. http://www.pbs.org/frontlineworld/rough/2005/07/seeds_of_suicid.html

17. Gruère, G. et. al. " Bt Cotton and Farmer Suicides in India." International Food Policy Research Institute, October 2008. http://cdm15738.contentdm.oclc.org/utils/getfile/collection/p15738coll2/id/14501/filename/14502.pdf

18. "Labeling around the World." Just Label It Campaign, July 2015. http://www.justlabelit.org/right-to-know-center/labeling-around-the-world/

19. "Labels for GMO Foods Are a Bad Idea." Scientific American, August 2013. http://www.scientificamerican.com/article/labels-for-gmo-foods-are-a-bad-idea/

20. "A Decade of EU-Funded GMO Research." European Union, 2010. http://ec.europa.eu/research/biosociety/pdf/a_decade_of_eu-funded_gmo_research.pdf

21. Zimmer, C. "Chipotle Says Adios To GMOs, As Food Industry Strips Away Ingredients." NPR News, April 2015. http://www.npr.org/sections/thesalt/2015/04/27/402632212/chipotle-says-adios-to-gmos-as-food-industry-strips-away-ingredients

22. "Frequently Asked Questions on Genetically Modified Foods." WHO, 2015. http://www.who.int/foodsafety/areas_work/food-technology/faq-genetically-modified-food/en/

23. Ledford, H. "CRISPR, the Disruptor." Nature, June 2015. http://www.nature.com/news/crispr-the-disruptor-1.17673

24. Gurian-Sherman, D. "Are GMOs Worth the Trouble?" MIT Technology Review, March 2014. http://www.technologyreview.com/view/525931/are-gmos-worth-the-trouble/

25. Northoff, E. "2050: A third more mouths to feed." Food and Agriculture Organization of the United Nations, October 2009. http://www.fao.org/news/story/en/item/35571/icode/

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From Corgis to Corn: A Brief Look at the Long History of GMO Technology - Science in the News

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by Gabriel Rangel figures by Anna Maurer Summary: To date, scientists have engineered bacteria that produce medication-grade drugs, crops with built-in pesticides, and beagles that glow in the dark. While these are all relatively recent advances in scientific technology, humans have been altering the genetics of organisms for over 30,000 years.

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Pros Site: https://www.labiotech.eu/interview/crispr-therapeutics-genome-editing/

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Tony Ho, Head of Research and Development at CRISPR Therapeutics, offers his insights into the advantages of CRISPR gene editing over other technologies and what the future holds for the genome editing field.
For decades, gene editing required engineering DNA-cutting enzymes, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), for every specific genetic modification. The advent of CRISPR-Cas9, a gene-editing tool that only requires a custom guide RNA molecule to find its target, revolutionized the field.

CRISPR gene editing has quickly risen in popularity as a research tool because it makes genome editing much simpler and faster than ever before. The technology is also being researched as a potential treatment for a wide range of genetic diseases.

That is the case of CRISPR Therapeutics, a company that is running clinical trials testing the technology in two blood conditions as well as in cancer. Tony Ho, who leads research efforts at the company, shares his views on the potential of the technology and his expectations for the future of gene editing.

How have genome editing tools advanced in recent years?

Genome editing technologies have been developed at a rapid pace over the past decade. Within the past five years, a novel system has been developed based on fundamental research of bacterial systems' immunity to viral infections.

CRISPR-Cas9 is based on RNA-guided targeting and is much simpler and faster than earlier methods. The ease of design, together with the remarkable specificity, efficiency, and versatility of the CRISPR-Cas9 system have revolutionized the field of genome editing and enabled rapid development of novel therapies.

What are the benefits of using genome editing tools for therapeutic purposes?

The advent of programmable nucleases has accelerated the development of gene editing from concept to clinic. Current advances in genome editing tools allow us not only to target monogenic diseases but also polygenic diseases, such as cancer and diabetes. Genomic editing also provides a degree of precision not previously possible by other therapeutic approaches through its ability to target individual cell types.

It also allows us to build on synthetic biology [techniques] that confer novel abilities to the cells. With these abilities, we can develop potentially curative therapies that may not be possible using traditional modalities.

What are the advantages of CRISPR over other genome editing tools?

The CRISPR-Cas9 system can modify DNA with greater precision than existing technologies. An advantage the CRISPR-Cas9 system offers over other mutagenic techniques, like ZFN and TALEN, is its relative simplicity and versatility.

Additionally, CRISPR-Cas9 has the potential of simultaneous multiple loci editing, making the technology easier, more efficient, and more scalable compared to other genome editing technologies. The programmability of the CRISPR-Cas9 system allows rapid prototyping of different therapeutic approaches, which leads to faster development cycles compared to other genome editing approaches.

What are the main challenges and limitations of genome editing tools?

While manufacturing is considered a challenge for the field, it is not a fundamental limitation. Companies are committed to overcoming challenges in order to bring medicines to patients at scale.

Additionally, it's important to push the boundaries of what genome editing tools can offer to medicine. For example, it's critical to move the field beyond therapeutic areas of rare diseases and hematologic malignancies, and into common diseases, like cancer and diabetes, where there is the potential to continue to see a dramatic impact and benefit to patients.

How do you expect the genome editing field to develop in the future?

We expect to see increased enthusiasm for the gene editing field, particularly around CRISPR-Cas9, as it is a powerful platform that is facile and scalable. We believe the next generation of cell therapies will be driven by increased edits. We have published papers at oncology conferences that demonstrate that CRISPR-Cas9 can generate perfectly viable and enhanced CAR-T cells with 10 or more edits.

What do you expect CRISPR will achieve in the next decade?

CRISPR-Cas9 is a revolutionary tool that can change the future of medicine. At CRISPR Therapeutics, we have five programs in the clinic, led by our two hematology programs, where we have demonstrated that a one-time CRISPR-based treatment can result in a functional cure for patients with sickle cell disease and beta-thalassemia.

We also have three ongoing clinical trials for allogeneic CAR-T therapies targeting B-cell malignancies, multiple myeloma, and renal cell carcinoma. Excitingly, we showed positive topline data in patients with relapsed or refractory B-cell malignancies, which demonstrate the potential of CRISPR gene editing for the treatment of cancers. We believe a number of cancers could be cured with CRISPR-based treatments, and we could replace a number of diseased or defective organs with regenerative medicine.

There is a world full of possibilities for CRISPR applications and I am very hopeful about what the future can bring.

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The Promises of CRISPR Genome Editing in Biomedicine

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Tony Ho, Head of Research and Development at CRISPR Therapeutics, offers his insights into the advantages of CRISPR gene editing over other technologies and what the future holds for the genome editing field. For decades, gene editing required engineering DNA-cutting enzymes, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), for every specific genetic modification.

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Cons site:

https://www.frontiersin.org/articles/10.3389/fmed.2021.649896/full

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Department of Pathology, Tongde Hospital of Zhejiang Province, Hangzhou, China

Department of Otolaryngology, Tongde Hospital of Zhejiang Province, Hangzhou, China

Department of Pharmacy, Tongde Hospital of Zhejiang Province, Hangzhou, China

Cancer is one of the most leading causes of mortalities worldwide. It is caused by the accumulation of genetic and epigenetic alterations in 2 types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. In recent years, development of the clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome engineering for different cancer research ranging for research ranging from fundamental science to translational medicine and precise cancer treatment. The CRISPR/CRISPR associated proteins (CRISPR/Cas) are prokaryote-derived genome editing systems that have enabled researchers to detect, image, manipulate and annotate specific DNA and RNA sequences in various types of living cells. The CRISPR/Cas systems have significant contributions to discovery of proto-oncogenes and TSGs, tumor cell epigenome normalization, targeted delivery, identification of drug resistance mechanisms, development of high-throughput genetic screening, tumor models establishment, and cancer immunotherapy and gene therapy in clinics. Robust technical improvements in CRISPR/Cas systems have shown a considerable degree of efficacy, specificity, and flexibility to target the specific locus in the genome for the desired applications. Recent developments in CRISPRs technology offers a significant hope of medical cure against cancer and other deadly diseases. Despite significant improvements in this field, several technical challenges need to be addressed, such as off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency, in vivo delivery of the Cas system components, and immune responses. This study aims to overview the recent technological advancements, preclinical and perspectives on clinical applications of CRISPR along with their advantages and limitations. Moreover, the potential applications of CRISPR/Cas in precise cancer tumor research, genetic, and other precise cancer treatments discussed.

Introduction

Cancer is one of the main causes of disease-associated mortalities worldwide with ever-increasing incidence worldwide ( 1). Comprehensive and large-scale sequencing databases have shown that genetic alterations, either specific to a certain type or common to several types, play crucial roles in tumorigenesis ( 2). Determining the structural and functional features of mutated genes, particularly long-tail molecular alterations, in genetic variations of cancer genomes play pivotal role in advancing cancer research ( 3, 4). However, systematic functional analysis of genes and mutations are time-consuming, expensive and laborious ( 5). Discovery of mutations that cause phenotypes relied either on random mutagenesis or indirectly on perturbation of transcripts by RNAi. The development of engineered nucleases such as zinc finger nucleases or transcription activator-like effector nucleases (TALENs) have made it possible to directly target and modify the genomic sequence ( 6, 7). Recently, genome engineering was greatly accelerated by the development of clustered regularly interspaced short palindromic repeats (CRISPR) technologies. Since the first use of CRISPR/CRISPR associated proteins (CRISPR/Cas) as a genome editing tool in 2013 in mammalian cells ( 8, 9), this toolbox has been extensively and continuously expanded. CRISPR/Cas systems are currently capable of not only manipulating the genomic sequence of cells and organisms, but also the introducing and site-specific targeting of epigenetic and transcriptional modifications ( 10- 12).

In the past decade, the emergence of the CRISPR technology has brought revolutionary advances into genome engineering and made it powerful tool in different cancer researches including fundamental sciences to translational medicine and precise cancer treatment. The CRISPR/Cas are prokaryote-derived genome editing systems that have shown promising contributions to detect, image, manipulate and annotate specific DNA and RNA sequences in various types of living cells. CRISPR/Cas, capable of specific genome modifications in living eukaryotic cells, making this technology one of the key scientific discoveries of the twenty-first century. The genomic modifications include; sequence deletions, insertions, substitutions, integrations, and epigenetic genes regulation. In the last few years, advancements in this technology make an ability to drive into both basic and clinical research applications. CRISPR/Cas system is an RNA-guided targeted genome engineering platform, attaining a considerable attention in experimental research, and revolutionize different fields of life sciences. Functionally, the CRISPR-Cas system is divided into 2 classes according to the structural composition of the effector genes. The class 1 CRISPR system consists of multi-subunits of effector nuclease complexes and includes the type I, III, and IV CRISPR systems. The class 2 consists of a single effector nuclease, and routine practice of genome editing has been achieved by the development of the Class 2 CRISPR-Cas system, which includes the type II, V, and VI CRISPR-Cas systems. Types II and V are utilized for DNA editing, and type VI for RNA editing. CRISPR techniques can induce both quantitative and qualitative changes in gene expression through the DSB repair pathway, transposase-dependent DNA integration, base editing, and gene regulation using the CRISPR-dCas or type VI CRISPR system.

The CRISPR systems were first observed in E. coli in 1987 ( 13) and then in several other bacteria species ( 14). The exact functions and roles of these short repeat sequences remained unclear until in 2005, when strong evidences have hypothesized that these repeated sequences function as a part of an adaptive immune system in bacteria. Several studies have reported the similarities between the phage DNA and these repeated sequences ( 15- 17). Further preclinical and animal model studies have demonstrated that CRISPR and CRISPR/Cas are associated to the adaptive immunity targeting foreign viral DNA ( 18). Mechanistically, two distinct RNAs including the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA) activate and guide Cas proteins to bind viral DNA sequences, which are subsequently cleaved together. The tracrRNA is a distinct type of RNA that interacts with the crRNA to produce the dual guide (g) RNA in CRISPR-Cas systems. The tracrRNA-crRNA interaction is pivotal for pre-crRNA processing, target recognition, and also cleavage.

CRISPR/Cas systems are adaptive (acquired) immune systems of prokaryotic and archaeal microorganisms and rely on ribonucleoprotein effector complexes. They eliminate invading phages, conjugative plasmids, and mobile genetic elements via reserving the memory of the encounters with foreign DNA in unique spacer sequences into CRISPR arrays ( 17- 19). Naturally, CRISPR systems integrate foreign DNA molecule into CRISPR arrays, which subsequently produce crRNAs, and containing protospacer regions that are complementary to antigenic invading DNA molecules, followed by hybridizing each crRNA with other non-coding tracrRNA. A crucial event, which forms a hybrid of crRNA-tracrRNA, and makes a complex with Cas nucleases that cleave target-DNA sequences nearby to short sequences called protospacer adjacent motifs (PAMs) ( 20, 21). Genetic engineers can manipulate the CRISPR/Cas system efficiently and can target genes of interest to regulate their functions effectively in any eukaryotic organism, particularly in mammalian. The molecular biology of the CRISPR/Cas reveals how it can be operated while using synthetic guide RNAs (gRNAs) and other components to the target region of interest in DNA molecule for the desired application and finds the disease-causing genetic variations ( 22). Typically, the most widely used CRISPR system i.e., CRISPR/Cas9 targets 5′ of a PAM sequence. They induce double-stranded breaks (DSBs), which can be repaired by 2 DNA repair pathways called, homology directed repair (HDR) and non-homologous end joining (NHEJ) ( 22). HDR pathway facilitates precise gene modifications in the presence of a repair template ( 23). However, in the absence of a repair template, DSBs repaired by the NHEJ pathway that introduces insertion or deletions by editing DNA region, resulting in target genes disruption by shifting the reading frame ( 23, 24).

CRISPR/Cas nucleases-induced DSBs are mostly repaired by efficient eukaryotic cellular NHEJ pathway rather than by the HDR ( 25). Meanwhile, utilizing the Cas9 nickases can optimize the yields of indel at the genes loci, and enhance the HDR efficiency ( 26). The efficacy of the HDR pathway can be improved by enhancing the HDR pathway via gene silencing or suppressing non-homologous end-joining proteins activity ( 23), using small-molecule reagents ( 23, 27), or expressed proteins ( 26- 28). Currently, DNA repair proteins have shown promising capacities in this regard, but in vivo implementation of these strategies are challenging. Moreover, DSBs in cells via DNA repair pathways are described that lead to many undesired genomic alterations, such as large deletions and translocations ( 29, 30). Various efforts have been made to improve HDR, such as DNA donor template designing, system delivery, and cell cycle synchronization ( 26, 31- 33).

Pros and Cons of CRISPR/Cas Technologies

In the last few years, advances in CRISPR/Cas technologies are spectacular and have shown considerable potential in several fields of life sciences research. CRISPR technologies are now considered more accurate, target-specific, easy to use, and multi-potential. Despite the remarkable advances in CRISPR, several limitations and concerns still exist, which need to be addressed and solved for the optimized Cas systems development. The current attempts at addressing all those concerns have been made to overcome these technical hurdles. In the following sections, the main limitations of the CRISPR technologies and recent advances to address them are discussed.

The off-target effects are still a major concern in complex eukaryotic organisms, most often in vivo for therapeutic applications ( 34, 35). The targeting specificity depends upon the gRNA of Cas9 and PAM sequences, and off-target cleavage in the genome ( 36). Different online editing programs have been developed and successfully utilized to identify and predict off-target cleavages in silico. However, these tools are limited to examining homologous genes and face shortcomings to predict, for example, epigenetic modifications. Technical advances like high throughput genome-wide next-generation sequencing, play an important role in reducing off-target effects ( 35, 37). Developing a well-optimized and engineered CRISPR system can significantly reduce the off-target effects. For instance, off-target effects can be reduced via increasing the nucleases cleavage specificity or reducing the time frame of functional activity for their applications. Different Cas proteins that exhibit enhancements in on-target specificity have been engineered that include eSpCas9, HF-Cas9, HypaCas9, and Sniper Cas9 ( 38- 41). Another approach is using Cas9 nickases, where one of the endonuclease domains were catalytically inactivated and as a result, the low off-target effect was analyzed in the genome ( 42, 43). Off-target effects induced by CRISPR can be reduced by limiting the duration of Cas9 activity. For example, the Cas9 system delivered via electroporation had shown a shorter half-life than delivered by other vector systems such as lentiviral or plasmid vector system-based cargo delivery methods. Dosage affects several parameters and the target specificity of cleavage can play an important role in their applications. Alternatively, the target specificity of Cas9 systems can be enhanced by direct modulation of the activity of the genome-editing proteins, Cas9 proteins, by reducing their activity following the target locus alteration ( 44). The Cas9 nucleases were activated by inserting a modified 4-hydroxytamoxifen-responsive intein, a cell-permeable small molecule, at specific positions in Cas9 ( 44). These conditionally active Cas9 systems could alter the target genomic sites and were reported to enhance the target specificity human cells, up to 25-folds higher than the wild-type Cas9 ( 44- 47).

Recent evidences have demonstrated that CRISPR system could be a highly efficient approach for the gene editing and manipulation applications in a variety of eukaryotic cells. However, HDR and indel mutation in some genome sites have shown low efficiency. To address the insufficient indel of Cas-system in the target sites, some efforts have been made to increase efficacy by either Cas engineering or gRNA ( 48, 49). The CRISPR/Cas proteins preceded DSB after the recognition of a PAM sequence ( 50, 51). Each type of Cas proteins contain their PAM sequence in the genome. Broadly speaking, type II CRISPR/Cas recognizes 3′ G-rich DNA sequences, while another type V, preferred 5′ T-rich sequences for their application.

The main issue in genome editing approaches is the unavailability of PAM in the desired gene loci. However, a range of Cas-nucleases variances such as SpCas9 and Cas12a are now available that are decreasing PAM restriction ( 52, 53). These kinds of advancements will provide flexibility in genome editing for the desired specific targets. In other ways, artificial intelligence plays a critical role and has been adopted for experimental designing to predict target sequences with high indel efficiency ( 54). The desired HDR efficiency to make genes functionally correct remains low, though different chemical and engineering tools have been used, i.e., chemical reagents, such as SCR7, NU7441, and KU0060648 ( 55, 56). The use of a donor template in the form of ssDNA led to increased HDR efficiency in cells ( 57). CRISPR/Cas often triggers cell apoptosis due to DSBs, rather than the desired genome editing ( 58). The safety issue raises when this genome editing system is utilized in human pluripotent stem cells (hPSCs). In response to DSBs by CRISPR, the activation of p53 occurred that triggers cellular apoptosis ( 59).

Recently, genome editing with base editors makes it possible to precisely fix desired targeted point mutations without requiring donor DNA templates, DSBs, or independence on HDR. In recent decades, these editing systems have been catalytically impaired nucleases, as a result, DSBs have not occurred. Importantly, 2 classes of base editors have been established; namely, cytosine base editors (CBEs) and adenine base editors (ABEs); they enable to catalyze the C*G base pairs (bp) conversion to T*A bp, and A*T bp to G*C bp, respectively ( 60- 62). Besides these, catalytically inactivated CRISPR-dCas9 (dCas9) was applied for epigenome modifications instead of a genome that can alter gene regulation. CRISPRa and CRISPRi system has been developed to activate and silence genes, respectively ( 63). For example, dCas9 in combination with histone deacetylase (HDAC), improved CRISPR system efficacy and optimal positioning and developed an organized system to study epigenome ( 64). The evaluation of off-target effects can be analyzed through several online bioinformatics tools to predict potential off-targets with similar sequences, such as CCTop ( https://crispr.cos.uniheidelberg.de), and Cas-OFFinder.

In addition, technical limitations and advances in the field of CRISPR technologies raise concerns for immunogenic toxicity. Recently, a study has shown that human subjects included, possessed pre-existing antibodies against Cas9. The obtained results showed that more than 50% of their subjects included in the study had immunity against the commonly used bacterial nucleases ( 65). In their study, the two extensively studied nucleases for gene therapy of Cas orthologs i.e., SaCas9 and SpCas9, were prevalent in human blood, and the human immune system has shown an immunogenic response against these nucleases. In this regard, extensive studies should be conducted particularly, for in vivo gene therapy applications. Furthermore, the gRNA triggers an innate immune response in human cells due to the presence of the phosphate group at the 5′ terminal ( 66). In addition, CRISPR has been extensively applied in clinical trials to modify somatic cells ex vivo, with the aim of reducing risk, and subsequently, transferring for in vivo gene therapy applications. However, the germ-line gene editing studies for therapeutic purposes still face ethical challenges. In this regard, the ongoing and near-future clinical trials on somatic CRISPR therapy need to be evaluated for the long-term to check the system efficacy and safety.

CRISPR Delivery Approaches and Challenges

An efficient delivery of both Cas9 and the single guide RNA (sgRNA) to the target cell is required for a successful in vivo administration of CRISPR/Cas9. The delivery approach should have high editing efficiency, induce low immunogenicity and deliver the Cas9/sgRNA specifically to the target organ or cell type. The first generation genome editing strategies in mammalian cells have been utilized the plasmid based expression of Cas9 and sgRNA ( 8, 9). Moreover, this approach is efficient for in vivo applications in model organisms such as mice because the plasmid can be delivered to the tissue by hydrodynamic injection ( 67) or electroporation ( 68- 70). However, in these applications the targeting delivery and editing efficiency are limited and control over the Cas9 activity is poor. Therefore, different viral and non-viral delivery strategies have been developed to enhance the performance of in vivo delivery of Cas9/sgRNA ( 71- 73).

Adeno-associated virus (AAV) vectors are effective and among the most common used viral vectors for gene therapy because of their unique features including non-integrating nature, high transduction efficiency and serologically compatible with most of human population ( 74- 77). Furthermore, the rich diversity of serotypes with distinct tissue tropisms enables AAVs to selectively target different organs ( 78, 79). However, the main limiting issue of AAVs for delivery of CRISPR and Cas9 is the limited cargo size of AAVs, so that the Cas systems and sgRNA should be encoded on additional separate vectors ( 74). AAVs can be administrated systemically or directly applied to the target organ for genome editing applications ( 75, 80- 82). Using lipid nanoparticles (LNPs) is an alternative approach to viral delivery, which offer availability, low cost and high compatibility ( 83- 85). LNPs have been employed for successful delivery of siRNA and mRNA in clinical trials ( 83, 86). Moreover, recent studies have demonstrated that LNPs can encapsulate and deliver the sgRNA and Cas9 mRNA to murine liver with high delivery efficiency and targeting performance ( 87- 90). Furthermore, multifunction and modified nanoparticles can be additionally loaded with a donor template and thereby allow homology directed repair ( 91). However, the nanoparticles based carriers with a donor template suffer low editing efficiency ( 91). The main focus of the current research is on improving and establishing CRISRP/Cas9 as a gene repair tool. However, it is expected that CRISPR/Cas9 would be translated into a therapeutic agent for cancer treatment in clinical setting. To achieve this goal, the main step is developing effective carriers for tissue-specific delivery of Cas9/sgRNA ( 92- 94).

Low editing efficiency in tumors and potential toxicity of the currently available delivery systems are the main limiting factors against translation of CRISPR/Cas9 technology into cancer therapeutics. The presence of an appropriate and effective alternative of delivery strategy is critical for CRISPR/Cas9 delivery, particularly where genome editing systems should be effectively conducted in the targeted organisms or cells. Until now, in vivo delivery of the Cas9 system remains challenging. Both physical techniques and viral vectors have been utilized for the delivery of the Cas9-based gene editing platform. The physical approaches are more feasible for in vitro delivery, but the viral vectors based techniques usually suffer limited packing capacities and poor safety profile. Recent preclinical and animal studies have demonstrated promising delivery performance and targeting efficacy of non-viral drug delivery systems such as polymeric and lipid nanocarriers for the delivery of CRISPR/Cas9 systems. These non-viral vectors are expected to be candidate carriers for the genome editing platform in the near future. The efforts in optimizing cationic nanocarriers with structural modification are described and promising non-viral vectors under clinical investigations are highlighted.

Different studies have recently developed a safe and effective strategy for antibody-targeted cell-specific delivery of mRNAs and siRNAs through systemically administration of LNPs ( 95- 97). In this regard, few studies have reported promising outcomes in using LNPs for the delivery of Cas9 mRNA and sgRNAs. The initial findings showed that aminoionizable LNPs could serve as a safe and efficient carrier for Cas9 components ( 87). Rosenblum et al. reported a single intracerebral injection of CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) into metastatic orthotopic glioblastoma enhanced the in vivo gene editing specificity up to ~70%, which inhibited tumor growth by 50%, induced tumor cell apoptosis, and enhanced survival by 30% ( 87). The cLNPs were engineered for antibody-targeted cell specific delivery to reach the distributed tumors ( 87, 88, 97, 98). Intraperitoneal injections of sgPLK1-cLNPs targeting EGFR improved the site specificity of gene editing in vivo by 80% for distributed ovarian tumors, and inhibited tumor growth, and increased survival by 80% ( 87). The capacity of disrupting gene expression in vivo in tumors is a promising feature for translating CRISPR tools into clinical applications and paves the way for developing gene editing techniques for cancer research and treatment and potential applications for targeted gene editing of non-malignant tissues.

Methods of Delivery

CRISPR technology has been reported one of the most promising therapeutic tool that could efficiently correct a variety of disease-associated mutations. In this view, it must be transported directly to their target site.

Multiple techniques have been developed for CRISPR delivery such as physical, viral, and non-viral delivery systems ( 99). Physical methods include microinjection, transfection, and electroporation that are most suitable for research purposes in cell culture. However, these strategies can be used for ex-vivo cell manipulation for adoptive transfer ( 100). Multiple studies revealed the delivery of Cas9 protein/gRNA ribonucleoprotein complexes into many cells of mammals by electroporation or transfection mediated by liposomes ( 101, 102). The findings of the studies have reported that the rate of insertion/deletion (InDel), induced by nuclease was 87% in induced pluripotent stem cells (iPSC). Furthermore, off-target cleavage was decreased, as compared with the transfection in plasmid DNA ( 102). Cas9/gRNAs delivered by lentiviral transduction or plasmid transfection have a longer half-life relative to Cas9-RNP complexes delivered through electroporation. Furthermore, Cas9-RNPs are active immediately post-delivery due to no lag, however, protein synthesis occurs. A reported study has been revealed that LNPs can efficiently deliver Cas9-RNP ( 71). Viral vectors, such as adenovirus, lentivirus, and adeno-associated virus (AAV) vectors have been used for delivery in clinical trials. Lentiviral vectors have been derived from HIV that provide stable and efficient delivery and can infect dividing as well as non-dividing cells, including the brain cells. Viral genes, such as vpr, vif, and nef are not needed for packaging. Therefore, the underlined genes are deleted while the expression of packaging genes are provided on separate plasmids to decrease the probabilities of reconstruction of wild-type virus ( 103). Moreover, lentiviral vectors are not suitable for therapeutic uses due to integration but this risk can be lowered via IDLV ( 104). Adenoviruses are viruses containing a linear double-stranded DNA genome of around 36 Kbp in length with four early and five late transcription units. The majority of the vectors are based upon adenovirus type 5 (Ad5). A recombinant virus has been constructed by removal of the early gene E1 or E1 plus E3 and grown in a packaging cell line that shows the expression of E1 to form infectious recombinant virus. Adenovirus can infect dividing as well as non-dividing cells and not show integration into the host genome ( 104).

Despite these applications, lentivirus and adenovirus vectors having some drawbacks, particularly safety problems associated with their immunogenicity ( 105). AAV vectors have significantly lower immunogenicity. AAV is a 4.7 Kb single-stranded DNA virus that needs E1 for the packaging of infectious viruses and can transduce dividing as well as non-dividing cells. In infected cells, the AAV genome can persist in an episomal form, but infrequently shows integration in the host genome. The most commonly used vectors for delivery of Cas9 are AAV because these vectors are very efficient and low immunogenic ( 106). However, the large size of the Cas9 endonuclease is a complication in its effective delivery with the gene for Streptococcus pyogenes Cas9 being about 4.2 Kb, while the size limit for AAV is between ~4.5 to 4.9Kb ( Figure 1). Ran et al. ( 108) described Cas9 orthologs and revealed that Streptococcus aureus (SaCas9) shows similar potency of editing to SpCas9, but is over 1 Kb shorter and can specifically and efficiently perform gene editing. In recent years, the isolation of another Cas9 ortholog has been carried out from Campylobacter jejuni, which is shorter with a size of 2.95 Kb ( 109). It has been revealed that the packaging of the CjCas9 gene could be performed in an AAV vector along with gRNA and a marker gene for the generation of high viral titers that may deliver more specific CjCas9, and was revealed to be a targeted endonuclease.

Figure 1. The CRISPR/Cas9 mechanism of action. Permission from ( 107).

CRISPR Applications in Cancer Research

Cancer is a disease of aberrant cell signaling that occurs due to a variety of genetic and epigenetic alterations in DNA. These alterations include the oncogenes, which enhance cell proliferation, and the tumor suppressors, which regulate cell growth and metabolism. The underline alterations lead to cancer progression. Nowadays, the ability of CRISPR to correct such cancer-associated alterations is an important objective for cancer diagnosis, cancer therapy, and other related applications. Hence, CRISPR is a promising tool that has been widely adopted in oncology research ( Figure 2) with focusing on; animal tumor model construction, the discovery of new drug targets; cancer gene therapy, genetic screening related to drug resistance, and many others. In the below section, some of the promising applications of CRISPR in cancer research are summarized.

Figure 2. Methods for delivery of Cas9-sgRNA complex to cell (A) Microinjection based delivery of Cas9-sgRNA (B) viral vector (AAV) based delivery (C) Lipofection (D) Cas9-sgRNA complex delivery into mammalian cells via Cell-penetrating peptides (CPP) revealed considerable genome editing with elevated level efficiency. With permission ( 107).

CRISPR for Tumor Research Modeling

Understanding complex mechanisms at the molecular level that drive tumor progression is a crucial step to advance therapeutics development. Usually, tumors arise due to multiple gene mutations, and this complexity makes it difficult for the development of full-pledge cancer models. In this view, the CRISPR system was considerably used to establish rapid tumor models, both in vitro and in vivo. These models allow identifying the genetic determinants and a comprehensive detail of the mechanisms that underlying tumor occurrence, progression, and development.

The generation of in vitro cancer model, while using CRISPR/Cas in mammalian cell lines with single or multiple gene(s) deletions is now easy and feasible ( 110), such as CRISPR-based mediated silencing of MELK, a cancer drug (OTS167) target in several clinical trials. The inactivation of MELK via CRISPR remains sensitive to OTS167 and does not affect the potency of cancer-derived cell lines. The underlined study explores the use of CRISPR that accelerate targeted cancer therapy research ( 111). Furthermore, CRISPR is applied to knock in or knock out functional alleles to develop drug resistance in vitro. CRISPR makes it possible to quickly evaluate candidate genes or specific mutations, associated with drug resistance ( 112). In this regard, a study was performed using CRISPR to identify mutations in crucial genes involved in therapeutic resistance that might be used for drug developmental strategies. For instance, NAMPT has been identified as the main drug target for the anti-cancer agent i.e., KPT-9274 ( 113). CRISPR, a versatile tool can be utilized to explore the genetic complexity of human cancer malignancies, such as myeloid malignancies, a malignancy that is driven by mutations in several genes, including Dnmt3a, Trp53, Tet2, Runx1, Ezh2, Smc3, Nf1, and Asxl1. By using the CRISPR system in single mouse hematopoietic stem cells, up to 5 genes were modified that induce the myeloid malignancies in mice ( 114). This study highlights the role of multiple gene mutations in cancer. CRISPR/Cas systems can be used to establish an in vivo tumor model. In exploring a complex mechanism of tumorigenesis, the in vivo cancer models play a critical role in the finding of key events i.e., pathogenesis and drug resistance. For example, the CRISPR system was used to attain mutations in important genes; P53, Kras, and Lkb1 in mice. These modifications/mutations in mice led to pathological changes in lung adenocarcinoma ( 115). The delivery of CRISPR cargoes into the living system plays an important role in model generation. For example, a lentiviral vector system used to deliver CRISPR into the desired target organs in vivo that create specific malignancy models ( Figure 3) ( 116, 117).

Figure 3. CRISPR/Cas systems applications in tumor research, drug development, and cancer therapies.

CRISPR-Based Screening Approaches in Cancer

CRISPR is effectively utilized to facilitate the discovery of next-generation targets or candidate genes that are sensitive or resistant to cancer therapy. Using the CRISPR system, several genetic screening studies were performed in vitro. It has been reported that in melanoma, the CRISPR library was applied to find the drug resistance mechanism of vemurafenib (PLX), an inhibitor of the BRAF protein kinase. In this study, new PLX-resistant candidates namely; TADA1, TADA2B, CUL3, and NF2 have been revealed ( 118). Moreover, using cell lines, the CRISPR screening has been utilized to identify oncogenes, and tumor suppressors in vivo ( 119, 120). The study showed the loss-of-function genetic screens and in vivo tumor mice models using CRISPR, and confirmed candidates gene and the pathways that are connected in the sensitivity and development of resistance to cancer immunotherapy ( 121). Strong evidences have highlighted the role of the Cas9 system in combination with RNA scaffolds that can be applied to induce site-specific epigenetic and transcriptional modifications while targeting a crucial region of the OCT4 promoters ( 122, 123). The OCT4 gene is recognized as one of the key players, which plays a critical role in tumorigenesis and therapy resistance.

Targeting Gene Regulation in Cancer

In cancer, gene regulation has affected both post-transcriptional and translational modifications that evolve cancer cells to survive and adapt within the microenvironment. For example; the Knockdown of micro RNAs (miRNAs) that enhance tumor initiation and development can prevent tumor occurrence, development, and anti-cancer therapy resistance. In this view, a study found a knocked-out miR-17 in colorectal cell line and injected into nude mice ( 124). The obtained results showed a stable gene-phenotype even after 2 weeks in tumor tissue which indicated that CRISPR can play a critical role in targeting miRNAs and can effectively target tumorigenic miRNAs. In cancers, abnormal expression of epigenetic regulatory genes plays an important role in tumorigenesis processes. Targeting acetyltransferase p300 (associated with a catalytic histone H3 lysine acetylation) using CRISPR system can activate gene promoters and co-regulatory components, which in turn facilitate the expression of the target gene and the associated genes ( 125).

Tumor Immuno-Regulation and Immunotherapy Approaches

Tumor immune escape is one of the key mechanisms of the cancer cell to survive and adapted in the tumor microenvironment, while the immune system unable to recognize it. Subsequently, tumor cells leash the immune cells through multiple pathways and thereby tumor cell progression and metastasis occur. Cancer immunotherapy is considered as an attractive strategy to target cancers and emerged as a potential therapeutic modality for the treatment of cancers. However, issues are existing to make it more precise for cancer patients ( 126, 127). In recent years, genetically engineered T cells against tumors have shown remarkable therapeutic effectiveness and performance. In human immune system, T cells play crucial roles in protecting the human body from infection by pathogens and eliminating mutant cells through specific recognition by T cell receptors (TCRs). Cancer immunotherapy utilizes the TCRs based recognition strategy to enhance the antitumor efficacy of T cells through releasing the inhibition of immune checkpoints and expanding adaptive immunity by promoting the adoptive transfer of genetically engineered T cells. T cells genetically equipped with chimeric antigen receptors (CARs) or TCRs have demonstrated significant effectiveness in treating different hematological disorders. However, the main issue of this approach is limited efficacy of engineered T cells in treating solid tumors. CRISPR system provides a new way to make engineered T-cells more efficient for the clinical treatment of different types of cancers ( 128). Moreover, the production of chimeric antigen receptor T (CAR-T) cells are significantly associated with the cancer therapy. Using CRISPR/Cas9, T-cells are genetically engineered in vitro, where the genes have been inserted and CAR protein have been expressed on the cell-surface that activated and recognized antigen on malignant cells very efficiently ( 129). Currently, several clinical trials are underway, using CRISPR for cancer immunotherapy applications ( clinicaltrials.gov). However, several efficacy and safety challenges still exist on using CRISPR/Cas for clinical applications.

Conclusion and Perspectives

The precipitous development in CRISPR technologies to their versatile and precise genome engineering in the last few years has been spectacular. These versatile tools now consider as an umbrella term, which revolutionized the life sciences and enabling advances in basic research for a variety of applications. It is believed that CRISPR can be established in clinics that can offer many therapeutic opportunities for treating human diseases, including cancer. Continued progress to improve and revolutionize new ways to deliver genome engineering tools into cells, and advance their capabilities to edit can implement these technologies for many therapeutic applications. CRISPR/Cas systems are widely utilized in tumor research for many applications both in vitro and in vivo models. Several clinical trials are currently underway, using the CRISPR/Cas system to accelerate or making the therapies more reliable to treat cancer effectively. However, extensive research work is still required to develop and applied these technologies in clinics. These technologies can provide wide-ranging opportunities for specific and desired genome engineering and can become a potent asset for the modern era of medicine. Continuous efforts to understand all their pitfalls, improving editing capabilities, and advances in the delivery systems will ensure the CRISPR system for the full potential to benefit society in near future.

Author Contributions

YY and LL: conceptualization, methodology, and writing-review and editing. YY, JX, SG, and LL: resources, data curation, and writing-original draft preparation. YY: supervision, project administration. YY and JX: funding acquisition. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Chinese Medical and Health Research Project of Zhejiang Province, China (Grant No. 2021ZB052 granted to YY and 2018ZT002 granted to JX).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

89. Miller JB, Zhang S, Kos P, Xiong H, Zhou K, Perelman SS, et al. Non-Viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew Chem Int Ed. (2017) 56:1059-63. doi: 10.1002/anie.201610209

108. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. (2015) 520:186-91. doi: 10.1038/nature14299

124. Chang H, Yi B, Ma R, Zhang X, Zhao H, Xi Y. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Sci Rep. (2016) 6:22312. doi: 10.1038/srep22312

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clustered regularly interspaced short palindromic repeats (crispr)

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CRISPR/Cas: Advances, Limitations, and Applications for Precision Cancer Research

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Cancer is one of the most leading causes of mortalities worldwide. It is caused by the accumulation of genetic and epigenetic alterations in 2 types of genes: tumor suppressor genes (TSGs) and proto-oncogenes.

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1."such alterations can change the organism's metabolism, growth rate, and/or response to external environmental factors."
2.It can increase toxin in the soil

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original site:
https://www.nature.com/articles/nature.2016.20988

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The move by Chinese scientists could spark a biomedical duel between China and the United States.

A Chinese group has become the first to inject a person with cells that contain genes edited using the revolutionary CRISPR-Cas9 technique.

On 28 October, a team led by oncologist Lu You at Sichuan University in Chengdu delivered the modified cells into a patient with aggressive lung cancer as part of a clinical trial at the West China Hospital, also in Chengdu.

Earlier clinical trials using cells edited with a different technique have excited clinicians. The introduction of CRISPR, which is simpler and more efficient than other techniques, will probably accelerate the race to get gene-edited cells into the clinic across the world, says Carl June, who specializes in immunotherapy at the University of Pennsylvania in Philadelphia and led one of the earlier studies.

"I think this is going to trigger 'Sputnik 2.0', a biomedical duel on progress between China and the United States, which is important since competition usually improves the end product," he says.

June is the scientific adviser for a planned US trial that will use CRISPR to target three genes in participants' cells, with the goal of treating various cancers. He expects the trial to start in early 2017. And in March 2017, a group at Peking University in Beijing hopes to start three clinical trials using CRISPR against bladder, prostate and renal-cell cancers. Those trials do not yet have approval or funding.

Lu's trial received ethical approval from a hospital review board in July. Injections into participants were supposed to begin in August but the date was pushed back, Lu says, because culturing and amplifying the cells took longer than expected and then the team ran into China's October holidays.

The researchers removed immune cells from the recipient's blood and then disabled a gene in them using CRISPR-Cas9, which combines a DNA-cutting enzyme with a molecular guide that can be programmed to tell the enzyme precisely where to cut. The disabled gene codes for the protein PD-1, which normally puts the brakes on a cell's immune response: cancers take advantage of that function to proliferate.

Lu's team then cultured the edited cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the edited cells will attack and defeat the cancer.

Lu says that the treatment went smoothly, and that the participant will get a second injection, but declined to give details because of patient confidentiality. The team plans to treat a total of ten people, who will each receive either two, three or four injections. It is primarily a safety trial, and participants will be monitored for six months to determine whether the injections are causing serious adverse effects. Lu's team will also watch them beyond that time to see if they seem to be benefiting from the treatment.

Other oncologists are excited about CRISPR's entry onto the cancer scene. "The technology to be able to do this is incredible," says Naiyer Rizvi of Columbia University Medical Center in New York City. Antonio Russo of Palermo University in Italy notes that antibodies that neutralize PD-1 have successfully put lung cancer in check, boding well for a CRISPR-enabled attack on the protein. "It's an exciting strategy," he says. "The rationale is strong."

But Rizvi questions whether this particular trial will succeed. The process of extracting, genetically modifying and multiplying cells is "a huge undertaking and not very scalable", he says. "Unless it shows a large gain in efficacy, it will be hard to justify moving forward." He doubts it will be superior to the use of antibodies, which can be expanded to unlimited quantities in the clinic. Lu says that this question is being evaluated in the trial, but that it's too early to say which approach is better.

About this article

Further reading

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CRISPR gene-editing tested in a person for the first time

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The move by Chinese scientists could spark a biomedical duel between China and the United States. A Chinese group has become the first to inject a person with cells that contain genes edited using the revolutionary CRISPR-Cas9 technique.

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Our ancestors didn't know much about genetic modification but they used selective breeding to breed the most helpful breed of dog to help they do things like manage crops and livestock.

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1.The bananas, created by an Australian scientist, contain a gene that is supposed to help people living in Africa produce vitamin A.
2.There are many experimental variations for expression and control of engineered genes that can be applied to minimize potential risks.

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The genetic modification helps the dogs hunt better than before and help see how it can change how the dogs hunt.

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In my Opinion bananas should not be Genetically modified. It decreases growth rate and toxins are soil. It also is very dangerous, you can not eat it because it can cause blindness and diseases.

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Less food would be wasted and would be used for other things

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we would be able to eat a whole watermelon.

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it would take longer to breed it with other watermelons.

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https://www.usatoday.com/story/news/nation/2015/01/13/iowa-trial-of-gmo-bananas-is-delayed/21678303/

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DES MOINES - A plan to have Iowa State University students eat genetically modified bananas has been delayed, apparently because of issues in shipping the fruit.

The bananas, created by an Australian scientist, contain a gene that is supposed to help people living in Africa produce vitamin A. Proponents say the gene came from a different type of banana and is safe to eat. But opponents contend the trial could expose volunteers to unknown dangers.

An ISU scientist had planned to feed the bananas to a dozen students during last fall's semester. But that didn't happen, a university spokeswoman confirmed last month. The spokeswoman said she didn't know why the trial was delayed or when or whether it would resume.

James Dale, the Australian scientist who developed the bananas, said in an e-mail to The Des Moines Register this weekend that he still hopes to complete the trial by midyear.

"Importantly, the nutrition study will go forward, but not until all of us are satisfied that the banana material meets quality standards," he wrote. "As you might imagine, given how you see bananas ripen in your own home, it has been a challenge shipping bananas from Australia to the U.S. and having them arrive in good condition."

Iowa State researchers sent an e-mail to students last summer seeking a dozen female volunteers for the study. Food science professor Wendy White said then that the volunteers would be paid $900 to eat the equivalent of three bananas each as part of a short-term, prescribed diet. Just one of the bananas would be the genetically modified type. Blood tests would be used to determine the body's reaction.

White said more than 500 students responded to the query, and 12 were selected.

The proposed trial has become a prominent topic in the long-running debate over genetically modified foods. Many scientists say such plants can safely include important nutrients, pest-prevention qualities or other attributes. But skeptics worry that genetically modified foods could be dangerous and uncontrollable, and they often portray the supporters as trying to quietly slip the products into public consumption.

Last month, the group African Centre for Biosafety decried the plan to eventually ship the genetically modified bananas to Uganda and other African countries. "Just because the GM banana has been developed in Australia and is being tested in the U.S. does not make it super!" Ugandan activist Bridget Mugambe wrote in a news release. "Ugandans know what is super because we have been eating homegrown GM-free bananas for centuries. This GM banana is an insult to our food, to our culture, to us a nation, and we strongly condemn it."

The group's director, Mariam Mayet of South Africa, said last week that she was unsure what to make of the delay in the trial or of Iowa State's lack of response to an open letter her group sent about the matter.

"How are we to interpret this silence? Disregard and disrespect? Dismissal perhaps?" Mayet wrote in an email to the Register. "... We would have expected not only a response, but greater transparency on the part of Iowa State - a willingness to share information and relevant and important bio-safety data pertaining to the trials."

She said she was puzzled by the claim that bananas are hard to transport, given how routinely they are shipped to grocery stores every day.

White said the goal of her research is to help people in Africa increase their production of vitamin A. "In Uganda and other African countries, vitamin A deficiency is a major contributor to deaths in childhood from infectious diseases," she wrote in a statement released by the university in July. "Wouldn't it be great if these bananas could prevent preschool kids from dying from diarrhea, malaria or measles?"

The scientist said the new type of banana includes a gene taken from another banana species, which naturally produces large amounts of beta-carotene. When people eat beta-carotene, their bodies turn it into vitamin A.

Residents of Uganda and nearby countries don't favor the type of sweet banana that naturally carries the extra beta-carotene, White said. So researchers have put the gene into a less-sweet type of banana that east Africans often use in cooking. White added that the new banana has no seeds, so there is no danger that the genetically modified plant would escape into nature.

Dale said Iowa State was chosen to conduct the trial because no independent Australian lab was available to do the work and analyze the results.

The Bill and Melinda Gates Foundation is supporting the project financially.

Jim Lorenzen, a senior program officer for the Gates Foundation, said the foundation expects the research to continue. "The Gates Foundation continues to support the Banana21 project, which is helping find ways to tackle vitamin A deficiency. We look forward to seeing the Iowa trial move forward after the project has completed the necessary due diligence," he wrote in a prepared statement sent to the Register.

Iowa State University has said that before the trial begins, details of how it would be conducted would be posted on a federal website, clinicaltrials.gov. As of Monday, the trial was not registered there.

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DES MOINES - A plan to have Iowa State University students eat genetically modified bananas has been delayed, apparently because of issues in shipping the fruit. The bananas, created by an Australian scientist, contain a gene that is supposed to help people living in Africa produce vitamin A.

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https://www.yourgenome.org/facts/what-is-a-gmo

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GMOs are organisms that have had their characteristics changed through the modification of their DNA.

GMO stands for genetically modified organism.

Genetically modified (GM) organisms are organisms that have had their genomes changed in a way that does not happen naturally.

By changing an organism's genome we can change its characteristics.

Any organism could be genetically modified, but laws restrict the creation of genetically modified humans, and the production and distribution of other GMOs is tightly regulated.

Common examples of GMOs are GM crops used in agriculture and GM model organisms used in medical research.

How do we make GMOs?

To create a GMO, we change specific characteristics by using lab techniques to delete or alter particular sections of DNA.

We can also change an organism's characteristics by introducing new pieces of DNA into their genomes. This could be:

DNA taken from the same species

DNA taken from a different species

DNA made synthetically in the lab.

The process of creating GMOs is called genetic modification or genetic engineering.

There are several techniques that can be used to modify a genome:

'Agrobacterium-mediated' genetic modification is a technique used to introduce new DNA into a plant genome using a modified microbe.

'Gene targeting' is a technique used to introduce new DNA into selected regions of a genome through a process called homologous recombination.

'Genome editing' is a technique used to change selected regions of a genome using enzymes designed to cut specific DNA sequences.

Why do we make GMOs?

GMOs are generally made for medical, environmental, or commercial reasons.

GM white mushrooms have had a gene that normally causes them to go brown altered so it no longer functions. These mushrooms take longer to go brown, prolonging their shelf-life.

GM bacteria have been developed that have had a gene for insulin added to their genome. These bacteria produce large quantities of insulin as they grow, which is then extracted and used by people with diabetes to control their blood sugar levels.

The Acer1 gene is thought to be involved in skin diseases like psoriasis. GM mice have been made where the Acer1 gene no longer functions to study what it normally does. These mice have hair loss and are less able to control heat and water loss from their skin.

What is not a GMO?

The genomes of organisms change naturally over time, for example:

when organisms mate, offspring get bits of DNA from both parents

mutations arise as a result of mistakes when DNA is copied

environmental factors like UV radiation can create changes in DNA.

These changes to the DNA are not 'genetic modifications' as they happen naturally.

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GMOs are organisms that have had their characteristics changed through the modification of their DNA. GMO stands for genetically modified organism. Genetically modified (GM) organisms are organisms that have had their genomes changed in a way that does not happen naturally. By changing an organism's genome we can change its characteristics.

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https://www.nature.com/scitable/topicpage/genetically-modified-organisms-gmos-transgenic-crops-and-732/

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Genetically Modified Organisms (GMOs) | Learn Science at Scitable

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If you could save lives by producing vaccines in genetically engineered bananas, would you? What if that meant exposing other organisms to foreign proteins and potentially upsetting the ecosystem? People have been altering the genomes of plants and animals through traditional breeding techniques for many years, but genetic engineering means we can now have more control over what changes are made, and we can also produce a wider range of variations. For example, we can incorporate genes from one species into a completely unrelated one. But where should we draw the line? The debate over large-scale commercialization and use of genetically modified organisms (GMOs), as well as products made from those organisms, has been growing ever since the advent of recombinant DNA technology.