Biotechnology: PCR, Gels and CRISPR

You can extract DNA from strawberries or split peas with kitchen supplies. Each ingredient has a job (see Learn.Genetics):

1. Mash and add detergent. Detergent breaks up the fatty membranes around the cell and the nucleus, releasing the DNA (see OpenStax Biology 17.1).
2. Add salt. Salt helps the DNA strands stick together.
3. Pour ice-cold alcohol on top. DNA does not dissolve in alcohol, so it comes out of solution as white, stringy clumps where the two layers meet. Keeping the alcohol cold gives a bigger yield.

Research labs follow the same basic plan with purer chemicals (see OpenStax Biology 17.1).

The polymerase chain reaction (PCR) makes many copies of one stretch of DNA in a test tube. A tube holds the DNA sample, two short primers that flank the target, the four DNA nucleotides and a heat-proof DNA polymerase called Taq, from the hot-spring bacterium Thermus aquaticus (see OpenStax Biology 17.1). A machine cycles the temperature:

1. Denature, about 95 °C: the two DNA strands separate.
2. Anneal, about 50 °C: the primers bind to their matching sequences.
3. Extend, 72 °C: Taq builds new strands starting from the primers.

Each cycle doubles the number of copies (see OpenStax Microbiology 12.2). The exact annealing temperature depends on the primers. Running 30 to 40 cycles gives more than a billion copies (see NHGRI PCR fact sheet). Kary Mullis shared the 1993 Nobel Prize in Chemistry for inventing PCR (see Nobel Prize).

In gel electrophoresis, DNA samples are loaded into wells at one end of a gel and an electric current is switched on. The phosphate groups in DNA carry a negative charge, so the DNA is pulled toward the positive electrode. The gel acts like a sieve: smaller fragments move through it faster, so they travel farther (see OpenStax Biology 17.1).

A ladder of fragments of known sizes runs in one lane. Comparing a sample's bands with the ladder estimates their sizes, measured in base pairs (bp) (see OpenStax Microbiology 12.2). Because people's DNA differs, cutting or copying the same regions gives different band patterns, which is how DNA can link a sample to a person or test for paternity (see OpenStax Biology 17.1).

Bacteria make restriction enzymes to defend themselves against foreign DNA, such as from viruses. Each enzyme cuts DNA at one particular short sequence. Many make staggered cuts that leave a few unpaired bases, called sticky ends. Any two pieces cut by the same enzyme have matching sticky ends, and the enzyme DNA ligase can seal them together (see OpenStax Biology 17.1).

A plasmid is a small circle of DNA that a bacterium copies on its own. Cut a plasmid and a human gene with the same enzyme, join them with ligase, put the plasmid back into bacteria, and every time the bacteria divide they copy the human gene too. A molecule joined together from different sources like this is recombinant DNA (see OpenStax Biology 17.1). Stanley Cohen, Herbert Boyer and colleagues first showed it working in 1973 (see Cohen et al.).

The first big payoff was medicine. In 1978, scientists at Genentech used bacteria to make human insulin, and in 1982 it went on sale as the first insulin made with recombinant DNA (see Quianzon & Cheikh).

Bacteria also defend themselves with CRISPR. They store snippets of viral DNA, make RNA copies, and use those RNAs to guide an enzyme such as Cas9 to cut the matching viral DNA (see MedlinePlus). In 2012, Martin Jinek, Emmanuelle Charpentier, Jennifer Doudna and colleagues showed that Cas9 can be steered by a single, designed guide RNA to cut any chosen DNA sequence that sits next to a short motif called the PAM (see Jinek et al.). Charpentier and Doudna shared the 2020 Nobel Prize in Chemistry (see Nobel Prize).

After the cut, the cell repairs its DNA. Researchers use that repair to delete or add a few bases or to swap in a new sequence. Most edits are made in somatic cells, the body's ordinary cells, and are not passed on to children (see MedlinePlus).

A first CRISPR medicine. Sickle cell disease is caused by a single-base mutation in a hemoglobin gene; the DNA and Genetics lesson shows how. In the treatment called Casgevy, doctors remove a patient's own blood stem cells, edit them with CRISPR-Cas9 in the lab, and return them. The edited cells make more fetal hemoglobin, which carries oxygen well (see FDA). The UK authorized Casgevy in November 2023, the first licensed medicine to use CRISPR (see MHRA), and the US Food and Drug Administration approved it for patients 12 and older in December 2023 (see FDA).

Genetic engineering has also changed farming. Three well-known examples:

• Flavr Savr tomato. The first genetically modified crop sold in stores, in 1994. A reversed copy of a gene slowed the softening of the fruit (see OpenStax Biology 17.1).
• Bt crops. These carry a gene from the bacterium Bacillus thuringiensis for a protein toxic to certain insects that eat the plant. The toxin is harmless to mammals, and organic farmers spray the bacteria themselves (see OpenStax Biology 17.1).
• Golden Rice. In 2000, researchers added genes that let rice grains make β-carotene, which the body turns into vitamin A, aimed at vitamin A deficiency (see Ye et al.).

In 2016 a committee of the US National Academies of Sciences, Engineering, and Medicine found no substantiated evidence that current genetically engineered crops pose more risk to human health than conventionally bred crops (see NASEM). The same report also described real concerns:

• Resistance. Some insects evolved damaging resistance to Bt where resistance-management plans were not followed, and weeds have evolved resistance to the herbicide glyphosate used with herbicide-tolerant crops.
• Ecology. The committee found no conclusive evidence of cause-and-effect environmental problems.
• Economics. Small farmers have often gained, but lasting benefits depend on institutional support.
• Labeling. Mandatory labels were not needed for food safety, but the committee said labeling involves social and economic values beyond safety.

These points are summarized in the report's news release (see NASEM news release).

The key line in debates over gene editing is between two kinds of cells. Editing somatic cells, as in Casgevy, affects only the patient. Editing the germline, meaning eggs, sperm or embryos, makes changes that can be passed down to future generations, and that is where most of the ethical debate centers (see NHGRI).

In 2018, He Jiankui announced the birth of twin girls from embryos he said he had edited (see Cyranoski & Ledford). The claim provoked an international outcry, and a Chinese court sentenced him to three years in prison for illegal medical practice (see Cyranoski).

Other questions apply even to approved treatments (see NHGRI):

• Consent. An embryo, and the generations after it, cannot agree to an edit made before birth.
• Safety. Many researchers argue germline editing should not be used to start pregnancies until it is shown to be safe. Around 2014, about 40 countries discouraged or banned germline editing research.
• Equity. New treatments may be open only to the wealthy and widen existing gaps in health care.

Each tool in this lesson rests on a discovery that came before it: isolating DNA, then cutting and joining it, then reading, copying and finally editing it.