Method of the Month: CRISPR-Cas9 Genome Editing
History of CRISPR-Cas9
During the 20th century, an avalanche of rapid advances in genetics and molecular biology finally uncovered the secrets of genetic inheritance. DNA was discovered to be the carrier of genetic information, and its double-helix structure was conclusively determined through x-ray crystallography. With this foundational knowledge in hand, scientists were able to design techniques of manipulating DNA and the genome, with organism-level consequences.
Paul Berg’s development of gene splicing technique in 1971 laid the groundwork for the first method of genetic engineering. In 1972, Herbert Boyer and Stanley Cohen created recombinant DNA plasmids and inserted them into bacteria. Their technique was not perfect, as they had no way of controlling where in the genome the recombinant DNA would be inserted. However, it represented a milestone in genetic science — for the first time, humans had redesigned an organism’s genome — and proved that it was possible to artificially transfer DNA between organisms.
By the early 21st century, scientists had a sound grasp of the molecular basis of genetic inheritance and how genomes could be sequenced. They also knew that since the structure of the DNA molecule is the same for most organisms, genes could be transferred between very different organisms, producing novel traits. However, they did not yet have a quick, precise, reliable way to remove and insert genes into any organism’s genome.
The search for such a method ended in 2012, when an international team of scientists led by Jennifer Doudna and Emmanuelle Charpentier developed a groundbreaking new method called CRISPR-Cas9 genome editing. This technology was so revolutionary that Doudna and Charpentier won the Nobel Prize for it in 2020, within a decade of its discovery.
CRISPR-Cas9’s basis: a bacterial immune system
CRISPR-Cas9 genome editing is based on a natural phenomenon first identified in E. coli bacteria. Bacteria are infected by a group of viruses called bacteriophages, and CRISPR is a component of the adaptive immune system that protects E. coli (and other bacteria) against infection by bacteriophages. Adaptive immunity is resistance to infection that is acquired when an organism survives an infection by a particular pathogen. Later, if the organism is re-infected by the same pathogen, its adaptive immunity will enable it to recognize and fight the infection, improving its chances of survival.
The acronym “CRISPR” stands for “Clustered Regularly-Interspaced Short Palindromic Repeats,” which are special DNA sequences found in bacterial genomes. These sequences consist of many short, identical segments of DNA that are palindromic (their sequence is identical when read from right-to-left and from left-to-right).
The CRISPR segments are separated from each other by unique sections of spacer DNA, which comes from bacteriophages that have previously tried to infect the bacterium or one of its ancestors (since CRISPR segments are part of a bacterium’s permanent genetic heritage). The first time a particular type of bacteriophage injects its DNA into a bacterium, the bacterium may be able to save itself by using an enzyme to cut the bacteriophage DNA into little pieces, destroying it. If this occurs, the bacterium inserts some of the broken pieces of bacteriophage DNA into its genome as spacer DNA between the CRISPRs. These pieces of foreign DNA in the bacterium’s genome are its “memory” for that particular type of bacteriophage, enabling it to rapidly destroy the bacteriophage if it is infected a second time.
In the future, a bacteriophage of the same type may attack the bacterium, injecting its DNA into the bacterial cytoplasm. When this occurs, the bacterium responds by transcribing the spacer DNA to make crRNA, a piece of RNA that is complementary to the bacteriophage DNA. The bacterium also transcribes and translates a gene for a Cas protein, an enzyme that can destroy foreign nucleic acids. The crRNA associates with the Cas protein, and the Cas-crRNA complex then binds the complementary sequence of bacteriophage DNA. This enables the Cas protein to cut up the bacteriophage DNA, destroying it.
Harnessing the CRISPR system for genome editing
The CRISPR-Cas9 genome editing system adapts the molecular structures in this simple bacterial immune system to add and remove genes in any organism’s genome. The steps involved are as follows:
First, a target location is chosen somewhere in the genome of the organism that is going to be genetically modified. This target location is where the Cas protein will create a double-stranded break. The target sequence should be approximately 20 nucleotides long, and should be unique (not repeated anywhere else in the organism’s genome). It also must have a small sequence called PAM adjacent to it, since Cas will not cut unless this sequence is present.
Second, a guide RNA (gRNA) is designed. The gRNA is analogous to the crRNA in E. coli’s CRISPR immune system. It is a piece of RNA that is complementary to the target DNA sequence. The traditional method of making gRNA is isolating a sample of the target DNA sequence, inserting it into a bacterial cell as a plasmid, allowing the bacterium’s RNA polymerase to transcribe the DNA into RNA, and isolating the RNA. However, it is now more common for the gRNA to be chemically synthesized.
Third, the gRNA must be combined with another small RNA. The gRNA alone has no affinity for the Cas9 protein, so it must be fused to a tracrRNA, which will anchor it to the Cas9 protein. The combination of gRNA and tracrRNA is called a sgRNA.
Fourth, the sgRNA is bound to a Cas9 protein (a specific type of Cas protein that Doudna and Charpentier isolated from Streptococcus pyogenes).
Fifth, the Cas9-sgRNA complex is inserted into the cell of the organism that is to be genetically modified. A common method of doing this is electroporation (using an electric current to create small holes in the cell membrane, through which the complex can be introduced).
Once in the cell’s nucleus, the sgRNA will scan the entire genome and help Cas9 locate the target sequence. The Cas9 protein will not bind the target site unless it recognizes the adjacent PAM sequence.
If the PAM sequence is present, Cas9 will act like a pair of scissors, cutting the target sequence at a location several nucleotides upstream of the PAM sequence. This double-stranded break creates two loose ends.
Sixth, a new gene is inserted into the nucleus via electroporation. This gene must be modified beforehand based on the structure of the target sequence: the ends of the new gene must resemble the sequences of the DNA that the loose ends were attached to.
When the new gene enters the nucleus, it will insert itself between the loose ends of the DNA, fixing the break caused by Cas9. This process is called homology-directed repair, since the ends of the inserted gene are homologous (very similar to) the sequences that the loose ends were previously attached to.
The end result of CRISPR-Cas9 gene editing is the establishment of a new gene at a very precise location in the organism’s genome!
Ethics of CRISPR-Cas9 Genome Editing
CRISPR-Cas9 technology has a myriad of practical uses and has been frequently used in the past decade to create novel solutions to biological problems. In one such instance from 2020, a team of scientists at UC Davis published a paper on their use of CRISPR-Cas9 technology to address a widespread population health concern. These researchers wanted to help mitigate vitamin A deficiencies in low-income countries, such as the Philippines. They used CRISPR-Cas9 gene editing to place a gene for beta-carotene (a compound that the human body converts into vitamin A) into rice plants. If this new genetically modified rice becomes the main variety cultivated in low-income countries, worldwide cases of vitamin A deficiency should decrease dramatically.
Despite conferring such enormous benefits to humanity, CRISPR-Cas9 genome editing also has a high potential for abuse. Some applications of CRISPR technology, such as editing human genomes, are at best ethically questionable and at worst morally reprehensible. While CRISPR can be used to save lives and improve population health, it can also be used to redesign organisms and change life as we know it. Such far-reaching consequences make it imperative that we carefully consider the ethical implications of each new application of this technology.
Bibliography
Artzt, Karen. “Mammalian Developmental Genetics in the Twentieth Century.” Genetics, vol. 192, no. 4, 2012, doi:10.1534/genetics.112.146191.
Dong, Oliver, et.al. “Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.” Nature Communications, vol. 11, no. 1178, 2020. doi.org/10.1038/s41467–020–14981-y.
Gostimskaya, Irina. “CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing.” Biochemistry, vol. 87, no. 8, 2022, doi:10.1134/S0006297922080090.
Thurtle-Schmidt, Deborah M. and Lo, Te-Wen. “Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates.” Biochemistry and Molecular Biology Education, vol. 46, no. 2, 2018, doi.org/10.1002/bmb.21108.
