Written Assignment 2

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Organisms, cells, and their molecules can be modified and engineered to achieve practical benefits for humans. Researchers who participate in this endeavor are part of the field known as biotechnology, which has had some remarkable successes and generated exciting possibilities—but also has some potential pitfalls. When you hear about biotechnology in the media, it is important to be cautious in distinguishing successes from promise. You’ve probably seen intriguing Advertisements for products that promise insights into the specific genes you carry and what they have to say about your health and ancestry. Or you may have read popular media stories highlighting attention-grabbing promises of biotechnological research, such as the following: • A kit that can test for cancer with just a drop of saliva • A device that can stimulate regeneration of neurons following spinal injuries • Targeted delivery of genetically engineered cells that can cure inherited diseases • “Gene chips” that can identify your risk of developing any one of hundreds of diseases and highlight treatments personalized for your genome The achievements made in agriculture, human health, and forensic science are particularly notable (FIGURE 7-1).1. Agriculture. The development of crops resistant to pests and disease has increased yields dramatically while reducing costs and, in many cases, reducing the amount of pesticides that must be used. Additionally, biotechnology can be used to produce foods with enhanced nutrition.2. Human health. Biotechnology has led to some notable successes in treating diseases, and to improvements in diagnosing and screening for genetic diseases.3. Forensic science. Forensic biotechnologies have led to spectacular advances in the capabilities of law enforcement and improvements in the criminal justice system. And per-haps equally important, the lessons we’re learning from these sobering cases are helping us to reform the criminal justice system. 3

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How would you create a plant resistant to being eaten by insects? Or a colony of bacteria that can produce human insulin? Surprisingly, although there are many different uses of biotechnology, a relatively small number of processes and tools are employed. These enable researchers to do the following: 1. Chop up the DNA from a donor organism that exhibits the trait of interest. 2. Amplify the small amount of DNA into larger quantities. 3. Insert pieces of the DNA into bacterial cells or viruses. 4. Grow separate colonies of the bacteria or viruses, each of which contains a different inserted piece of donor DNA.

Now let’s explore each step in more detail: Tool 1. Chopping up DNA from a donor organism To begin, researchers select an organism that has a desirable trait. For instance, the researchers might want to produce human growth hormone in large amounts. Their first step would be to obtain human DNA and cut the DNA into smaller pieces. Cutting DNA into small pieces is a process that is done by restriction enzymes. Restriction enzymes have a single function: when they encounter DNA, they cut it into small pieces. These enzymes evolved to protect bacteria from attack by viruses. Upon encountering DNA from an invading virus, a restriction enzyme recognizes and binds to a particular sequence of four to eight bases on the invader’s DNA and cuts there, thus making it impossible for the virus to reproduce within the bacterial cell. Since all DNA has the same structure, these enzymes can cut DNA from any source as long as the specific four-eight base sequence is present (bacteria protect their own DNA by modifying it, or it would be cut up also). Dozens of different restriction enzymes exist, each of which cuts DNA at a different location.

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Tool 2: Amplify In biotechnology, it is common for only a small sample of the DNA of interest to be available. For this reason, the polymerase chain reaction (PCR) is a valuable tool for researchers. PCR is a laboratory technique that allows a tiny piece of DNA to be duplicated repeatedly. The process involves briefly heating a solution containing the DNA of interest, which causes the two DNA strands to separate. As the solution cools, an enzyme covalently bonds free nucleotides with their complementary bases on each of the separated single strands of DNA. Adding primers with sequences complementary to the region that is desired amplified makes it possible to selectively amplify regions of interest. The result is two complete double-stranded copies of the DNA, and the process can be repeated again and again until there are billions of identical copies of the original sequence.

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Tool 3: Insert Inserting foreign DNA into a target organism, a process known as gene editing (or genome editing), alters an organism’s genome. In the human growth hormone example, the researchers might want to transfer the human growth hormone gene into the bacterium Escherichia coli, creating transgenic organisms(that is, organisms with DNA inserted from a different species). The bacteria can then produce large amounts of the desired product. To create a transgenic organism, researchers must physically deliver the DNA from a donor species into the recipient organism. This delivery requires a “vector”—something to carry the donor DNA—and is often accomplished using plasmids, circular pieces of DNA that can be incorporated into a bacterium’s genome (FIGURE 7-4).A restriction enzyme recognizes and binds to a particular sequence of four to eight bases of the plasmid and cuts there, thus allowing insertion of the matching sequence of donor DNA (matching because it was cut by the same restriction enzyme). After insertion of the plasmid into the bacterial cell, genes on the plasmid can be expressed in the bacterium and are replicated whenever the cell divides, so both of the new cells contain the plasmid with the donor gene. In other cases, genes are incorporated into viruses instead of plasmids. The viruses can then be used to infect organisms and transfer the genes of interest into those organisms.

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Tool 4: Grow Once a piece of foreign DNA has been transferred to a bacterial cell, every time the bacterium divides, it creates a clone, a genetically identical cell that contains the inserted DNA. With numerous rounds of cell division, it is possible to produce a huge number of clones, all of which transcribe and translate the gene of interest. In a typical recombinant DNA experiment—that is, one in which DNA from two or more sources is brought together—a large amount of DNA may be chopped up with restriction enzymes, incorporated into plasmids, and introduced into bacterial cells. The bacteria are then allowed to divide repeatedly, with each bacterial cell producing a clone of the foreign DNA fragment it carries. Together, all of the different cells containing all of the different fragments of the original DNA are called a clone library or a gene library (FIGURE 7-5). Researchers can later identify those bacteria with the gene of interest and grow them in large numbers.

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Since 2012, one technology has generated almost unprecedented enthusiasm while racking up some impressive successes in delivering on its early promise. It goes by the acronym CRISPR (pronounced “crisper”) and is a system for editing DNA. What makes CRISPR noteworthy is that it brings much greater precision and efficiency to gene editing, acting as a cut-and-paste tool that enables researchers to modify almost any gene in any organism. CRISPR stands for clustered regularly interspaced short palindromic repeats. The name describes the organization of DNA that originally came from viruses but regularly is incorporated by bacteria within their own genomes to serve as a sort of immune system that can help thwart infection by potentially lethal viruses.

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Adapting the CRISPR system for use in biotechnology is straightforward: 1. After identifying a particular DNA sequence of interest (perhaps to fix or inactivate a defective gene), researchers synthesize an RNA molecule—the “guide” molecule—with a sequence that matches the target gene to be sliced (following the complementary base-pairing rules described in Section 6-2). 2. The DNA sequences for the CRISPR RNA and the Cas9 enzyme are introduced to the target cells, using a plasmid as vector. 3. Within the target (host) cell, the RNA leads Cas9 to exactly the desired location on the cell’s DNA, and the enzyme cuts the DNA there. 4. At that location, a sequence can then be inserted that repairs, or alters in some other way, the host cell’s DNA.

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CRISPR is considered a breakthrough in biotechnology because the ability to target and snip DNA precisely at a specific sequence opens the door to changing an organism’s genes in almost any way imaginable. Just a few of the initial successful uses of CRISPR reveal the wide variety of potential applications (Figure 7-7). Researchers have achieved the following: • Increasing significantly the muscle mass in the legs of a dog • Inactivating immune system markers on tissue from a pig—by editing 62 genes simultaneously—in a way that could facilitate use of transplants from pigs into humans • Introducing mutations into human stem cells to produce tissues with disease properties that can serve as model systems for studying common human diseases. There is even more enthusiasm surrounding CRISPR’s potential for fighting some of the most harmful diseases that affect humans. Significant work has already been done with CRISPR in altering the biochemistry of mosquitoes so that they cannot host or transmit the parasite that causes malaria. Eradicating malaria could save half a million lives every year. With CRISPR, it might also be possible to inactivate the genes in disease-causing bacteria that confer resistance to antibiotics. Or to target invasive species that damage environments and threaten native species.

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Without question, CRISPR has enormous potential. But some concerns (and other issues) remain. For starters, numerous legal proceedings are under way as several universities and companies fight over who invented the valuable techniques and has the rights to develop and profit from them. And perhaps a more significant impediment to unrestrained exploration of the possible applications of CRISPR relates to ethical issues. These include concern about the potential for editing human embryos or germline cells (sperm or eggs), because the gene changes could be passed to subsequent generations. Critics have also noted that the consequences of introducing new or altered genes into the genomes of natural populations of organisms are difficult to predict. Even some desirable outcomes—eradicating the mosquito populations that transmit malaria, for example—might have secondary, harmful effects, such as for bird or bat populations that rely on mosquitoes as a food source.

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