Gene Editing Using CRISPR -- Why the Excitement?

JAMA Viewpoint
Gene Editing Using CRISPR -- Why the Excitement? Anthony L. Komaroff, MD
JAMA. Published online August 10, 2017. doi:10.1001/jama.2017.10159
jamanetwork.com/journals/jama/fullarticle/2646800

The gene-editing technique known as CRISPR (clustered regularly interspaced short palindromic repeats) is only 5 years old, yet it has galvanized biomedical research and raised important ethical questions. What is it, how does it work, and how could it change medical practice?
The Evolution of Gene Editing

Biomedical scientists have been “editing” (or, at least, altering) genes for many years. Recombinant DNA technology allowed particular genes to be inserted into a plasmid (a circle of DNA) or into a virus: bacterial and yeast cells now could produce therapeutically useful human proteins, and viral vectors could perform gene therapy in humans. Gene targeting and RNA interference allowed the knockout of particular genes and the insertion of a healthy gene at the site of a defective gene. Zinc finger proteins and transcription activator-like effector nucleases (TALENs) precisely altered specific genes. Then came CRISPR. Compared with these previous technologies, CRISPR is easier, faster, less expensive, and more powerful.


How Does CRISPR Work?

CRISPR technology depends on the fact that a strand of nucleic acid (DNA or RNA) with a particular sequence of bases binds naturally to another nucleic acid strand with a matching (complementary) sequence. The workhorse of CRISPR technology is a complex of RNA and protein (primarily nucleases). The most widely used complex is called CRISPR-Cas9.

The RNA in a CRISPR-Cas9 complex has 2 functions. One part is programmed to recognize a particular sequence of bases in the target gene, while the other holds the Cas9 proteins close. Cas9 then unwinds or unzips a double helix so that the target nucleic acid sequence is made “visible” to the matching CRISPR RNA sequence, which quickly binds to its target. Then Cas9 cuts both strands of the target DNA precisely at the right spot.

CRISPR can affect the structure of a gene and can correct a single-base mutation. For example, CRISPR can transform the gene for hemoglobin S (sickle cell globin) into the gene for hemoglobin A. It does this by adding into the CRISPR mix a short DNA sequence for the healthy hemoglobin A gene: after Cas9 cuts the globin gene at the point of the sickle mutation, the sequence that codes for hemoglobin A is inserted.

CRISPR also can affect the expression of a gene: it can shut off the production of a protein, or ramp it up. For example, it can edit the messenger RNA made by a gene; alternatively, it can edit the “noncoding” DNA in the genome that controls the expression of specific genes.
Using CRISPR in Living Organisms

The DNA that codes for the CRISPR RNA, and for the CRISPR proteins (such as Cas9), can be introduced into living organisms using viral vectors, lipid nanoparticles, and other means. Typically, the goal is to reach just the pertinent cells—for example, just hematopoietic stem cells if the goal is to generate hemoglobin A instead of hemoglobin S.

The delivery of CRISPR to a target tissue can be hazardous, and the CRISPR payload does not always reach the intended destination. One way of solving that problem is to first edit an organism’s target cells (such as hematopoietic stem cells) in the laboratory, allow the edited cells to multiply, and then reinfuse those edited cells into the organism, where they home to their target tissue (such as the bone marrow).

CRISPR also can generate organisms in which every cell has been altered in a specific way. For instance, editing the genes in a fertilized egg leads to animals that have the edited genes in every cell.


How Was CRISPR Discovered?

Some medical breakthroughs are inventions, others are discoveries, and some, like CRISPR, are both.

The path leading to CRISPR was tortuous and full of surprises. First, scientists discovered unusual structures in bacterial genomes: clustered regularly interspaced short palindromic repeats (CRISPR) followed by genes for various nuclease proteins. These structures were found to somehow aid a bacterium’s ability to prevent infection by viruses (bacteriophages). The mechanism was subsequently discovered: the CRISPR genes edit viral genes, thereby disabling the viruses.1,2

Then came ultimate discovery and the invention. Scientists realized that the CRISPR-Cas9 complex could be modified and simplified to produce a programmable tool by which the nucleic acids of all plant and animal species could be precisely edited.1,2 The technology was optimized for use in mammalian cells and to edit multiple genes simultaneously.3,4 Subsequent variants of CRISPR technology involving different nuclease proteins have been developed to make CRISPR simpler and more precise.
Limitations of CRISPR

CRISPR allows remarkably precise editing, but it is not perfect. Sometimes, unintended nucleic acid sequences get edited—so-called off-target effects. This may occur more frequently than had been thought. Furthermore, some genetic “defects” that increase the risk of one disease decrease the risk of another. For example, people with sickle cell disease are less vulnerable to malaria: fixing hemoglobin S to solve one problem may cause another. Moreover, scientists understand relatively little about the health effects of “fixing” any particular polymorphic variant. In addition, like any powerful technology, CRISPR could be abused. Indeed, the US intelligence community has publicly expressed concern that CRISPR could produce a weapon of mass destruction.5


How Is CRISPR Affecting Biological Research?

CRISPR technology has transformed genetic research with plants and microbes. It has greatly aided research with many animal models, such as fruit flies, worms, and zebrafish, and has revolutionized the process for creating genetically modified mice,6 essential tools for medical research. Also, CRISPR has made possible the editing of primate genomes. Moreover, CRISPR has been used to treat disease in mice: a viral vector carried a CRISPR complex programmed to edit a mutated gene for dystrophin into the skeletal and cardiac muscle of mice with Duchenne-like muscular dystrophy, and a single treatment led to greatly improved muscle function.7


How Might CRISPR Affect Medical Practice?

CRISPR has greatly reduced the tumor burden of human prostate cancer cells in mouse xenografts.8 It has made possible a test that can be used in resource-limited settings for immediate diagnostics, such as for Zika virus.9 CRISPR also has been used for editing genes in T cells, ex vivo, to program them to attack a patient’s tumor when those cells are reinfused.

Similarly, it may be possible to edit a patient’s hematopoietic stem cells to correct sickle cell anemia and β-thalassemia. Using CRISPR in humans to edit solid-organ cells in vivo, however, as in the mice with muscular dystrophy, is further off.

The role of CRISPR in treating most of the major causes of disability and death in the developed nations is uncertain. These diseases typically are influenced by variants in multiple genes, each of which only slightly increases disease risk. More important, lifestyle modification is likely to have a greater influence on preventing and controlling these diseases than gene editing, no matter how powerful the technology becomes.

Experiments using CRISPR to edit human germ cells are subject to particular scrutiny because such experiments affect all descendants. Gene editing followed by in vitro fertilization could eliminate the risk that a conceptus would inherit (and pass on) a terrible disease—but it also could create an editing error that would harm future generations. The use of CRISPR to edit disease-related genes in germ cells also could generate a demand to protect all offspring against future risks (such as reducing the risk of Alzheimer disease by converting the ε4 variant of APOE to an alternative polymorphism). Moreover, CRISPR could create a demand for “designer babies” with certain desired traits. Society needs to grapple now with the ethical questions raised by such demands, before the technology can satisfy them.
How Might CRISPR Affect Human Health More Broadly?

CRISPR is being used to make plants and animals resistant to disease; to create certain animals (eg, pigs and cows) that become “bioreactors” for making therapeutic human proteins; and to generate pigs that could serve as human organ donors (such as for heart valve tissue), because their organs do not elicit an immune attack following transplantation. Theoretically, CRISPR gene drives could render mosquitoes all over the globe incapable of hosting various human pathogens.

The discovery and invention of CRISPR are already having a profound effect on biomedical research and are beginning to have an impact on medical practice. The scientists who created the CRISPR technology probably cannot imagine all of the ways in which it will be used. Two of those scientists said it best: “Every time we unlock one of nature’s secrets, it signals the end of one experiment—and the beginning of many others.”5

References at the original source.