CRISPR pioneers recognized with the 2020 Nobel Prize in Chemistry
Congratulations to Jennifer Doudna, Ph.D., and Emmanuelle Charpentier, Ph.D. for winning the 2020 Nobel Prize in Chemistry for the discovery of CRISPR- gene editing mechanism, also known as CRISPR/Cas9.
In a seminal 2012 paper, these Nobel laureates showed that the key components of the ancient immune system of bacteria and archaea could be retooled to edit genomes of any organism including humans with extremely high precision. And since then, CRISPR-Cas9 technology has transformed the life sciences making gene editing common in laboratories around the world.
Let’s look at their discovery in detail and understand what was so special about it that made them win this prestigious award.
Starting with the basics, gene editing aims to rewrite the genetic code, in order, to treat a genetic disease or to add desirable characteristics in an organism.
Genes are the segments of DNA that contain a specific sequence of As, Gs, Cs, and Ts and that direct the cell to produce proteins, which perform a vast array of functions in the body. Any change in the DNA sequence of these genes is called a mutation. Mutations in the genes can cause changes in the messenger RNA, which can further lead to the production of defective protein or no protein at all.
To understand it better, let’s take the example of sickle cell disease. Sickle cell disease occurs because of the mutation in the HBB genes in the seventeenth position. In this mutation, the A nucleotide is replaced by the T nucleotide. When translated into amino acids, this mutation results in the replacement of amino acid glutamate by amino acid valine in the beta-globin subunit of the hemoglobin protein. This mutation of a single nucleotide in hemoglobin causes the hemoglobin molecules to stick together and form abnormal filaments that change the shape of the red blood cells eventually leading to anemia, which can cause an increased risk of stroke and infection, and severe bone pain.
In this case, gene editing of the mutated HBB gene replaces the incorrect T nucleotide at position seventeenth with the correct nucleotide A, after which, the repaired gene translated into amino acids results in the functional beta-globin subunit of the hemoglobin protein. Thus, correcting the sickle cell disease permanently.
Although there are several tools available like ZFN, TALENs, etc. for gene editing, but over the past decade, the CRISPR-Cas9 system has become a very popular tool to edit the genome of any organism including humans. This is because, unlike other tools, CRISPR-Cas9 is fast, cheap, precise, and relatively easy to use.
CRISPR- Cas9 system has countless applications for gene editing, ranging from the treatment of genetic diseases like sickle cell disease, cystic fibrosis, etc. to the generation of engineered crops. CRISPR could, one day, even allow the scientists to wipe out the entire population of malaria-causing mosquitoes and not just this, it could also facilitate the resurrection of once-extinct species.
But the potentials and possibilities that CRISPR has opened go well beyond gene editing. One of the very recent and apt example of this is the development of CRISPR-based diagnostic kits to detect SARS-CoV-2 that causes COVID-19.
What is the CRISP-Cas9 system?
The CRISPR-Cas system was originally adapted from a naturally occurring defense system in bacteria. The roots of the discovery of CRISPR date back to 1987, when scientists noticed that the E.coli genome contains an unusual genetic structure composed of alternating repeat and non-repeat DNA sequences, whose biological significance was unclear at that time. The function of the CRISPR system was brought to light 20 years later in 2007 when scientists identified two different CRISPR loci in Streptococcus thermophilus strains. When the non-repeat DNA sequences also called spacers of the CRISPR loci were sequenced, it was found that the spacers were homologous to bacteriophage DNA and plasmid sequences. Thus leading to the hypothesis that CRISPR may be a defense mechanism of bacteria against foreign elements. And in 2012, the potential of CRISPR-Cas9 in gene editing was realized by Jennifer Doudna and Emmanuel Charpentier and their team.
CRISPR stands for Clustered Regularly-Interspaced Short Palindromic Repeats. The full form of CRISPR tells about 2 main parts found in CRISPR. First of all, there are repetitive sequences that are short segments of DNA, 20–40 bp in length, and also the repetitive sequences are palindromic. A palindrome is a sequence of letters that read the same either from the left or from the right. Like in never odd or even. In the case of palindromic repeat sequences, each repeat is arranged in a palindromic fashion, meaning that the repeat’s sequence on one strand is identical to the opposite strand’s sequence when both are read in their respective 5’ to 3’ direction. Because of the palindromic nature, the two halves of the sequence are complementary, allowing them to base pair. Thus, when these palindromic sequences are transcribed to form RNA, they are able to form hairpin shaped secondary structures.
Additionally, the repeat sequences are highly conserved within a CRISPR locus means that these repeat sequences within a CRISPR locus are all identical one after another after another and so on.
The full form of CRISPR Clustered Regularly-Interspaced Short Palindromic Repeats also suggests that the short palindromic repeat sequences are regularly interspaced. The spacer DNA is present between these repeat sequences. In this figure, the spacer DNA is shown by the colored boxes.
Spacer DNA, or simply referred to as spacers are not identical. Each segment of the spacer is unique. These unique sequences match the DNA found in the viruses. These spacer sequences are some kind of acquired immune system in bacteria that protect the bacteria from getting infected with viruses. These spacers are used by the bacteria as recognition elements to find matching virus genomes and destroy them. In other words, these spacers function as an immunological memory bank, storing sequences from previous encounters with invading organisms.
The CRISPR array also consists of CRISPR-associated genes, generally called cas genes. These cas genes make Cas proteins. The Cas proteins can be helicases, the proteins that unwind DNA, or the nucleases that cut the target viral DNA. The CRISPR immune system works through the cooperation of many diverse Cas proteins. And these Cas proteins constitute the backbone of the CRISPR system. Thus the system is generally called the CRISPR-Cas system.
Suppose the bacteriophage attacks the bacteria and injects its genetic material into the bacterial cell. After infection, the CRISPR locus is transcribed into a precursor-CRISPR RNA, and cas genes are translated to form Cas proteins including Cas9. The pre-crRNAs are processed to form mature crRNAs, each containing the spacer DNA.
The mature crRNAs then form complexes with a second RNA called tracrRNA and CRISPR associated protein Cas9 to form a complete search complex. These RNAs together are called guide RNA as they guide the Cas9 to interact with the viral DNA sequences matching the 20 nucleotides in the guide RNA. Thus allowing the Cas 9 to make a double-stranded break in the target viral DNA, eventually degrading the viral DNA and thus preventing the bacteria from viral infection.
But imagine if the phage invading the bacteria is new, and the bacteria don’t have any protospacer matching to the viral genome. In this case, a protein complex called Cas1 and Cas2 identifies the invading viral DNA and excises a segment of a specific length from the viral DNA. This excised segment of viral DNA is known as the protospacer. Then the protospacer is added into the front of the CRISPR array between the two repeat sequences. By this mechanism, bacteria generate the embedded memory of the invading virus.
Once the protospacer from the invading DNA is integrated into the loci of CRISPR, the crRNA containing the protospacer forms complexes with a second RNA called tracrRNA and CRISPR associated protein Cas9 to form a search complex. The search complex guides the Cas9 to interact with the viral DNA sequences matching the 20 nucleotides in the guide RNA. Thus allowing the Cas 9 to cleave the viral DNA, eventually preventing the bacteria from viral infection. So you can say that in bacteria, the CRISPR-Cas9 system evolved as a defense tool to degrade the invading viruses.
Potential of CRISPR-Cas9 in gene editing
The potential of CRISPR-Cas9 in gene editing was realized in 2012 by Jennifer Doudna and Emmanuel Charpentier and their team. The simplified engineered CRISPR-Cas9 system was developed by linking the two RNAs crRNA and tracrRNA to form a single guide RNA (sgRNA) or simply called guide RNA. The guide RNA consists of a 20 letter guide sequence that is complementary to a small sequence of target DNA of the host that is required to be edited. In the other words if the DNA has a sequence that reads 5’ ATGCGC then the RNA guide sequence is complementary and reads 3’-TUCGCG-5’. Because RNA contains U (uracil) instead of a T (thymidine).
Cas9 protein then attaches to the guide RNA and the whole complex then binds to the target DNA in the host genome. After the guide RNA binds at the desired position in the genome, the Cas9 enzyme introduces double-stranded breaks in the target DNA. Once the DNA is cut, researchers could use the cell’s own DNA repair machinery to add or delete pieces of genetic material, to make changes to the target DNA. The researchers published their findings in the journal Science in 2012.
After the discovery, gene editing by the CRISPR-Cas9 system is being explored in:
- Treatment of various diseases like sickle cell disease, hemophilia, cystic fibrosis, heart disease, cancer, etc.
- Generating gene drives to modify not just a single organism but an entire species, thus controlling the numbers of animal or insect species that transmit infectious diseases in humans. For instance, malaria-transmitting mosquitoes.
- The resurrection of once-extinct species.
- Editing genes of various crops to make them more nutritious, better survivors of heat and cold conditions, and resistant to pathogens.
- Creating designer babies, etc.
CRISPR-Cas system in diagnosing infectious diseases
After establishing the importance of the CRISPR-Cas system in gene editing, the scientists are harnessing this technology for diagnosing infectious diseases. A team of scientists at Broad Institute and MIT found that two cousins of Cas9 called Cas12 and Cas13 can be harnessed to actually detect human disease. While Cas12 targets DNA, Cas13 targets the RNA. Part of the mechanism of their action is similar to that of Cas9. Guide RNA which is complementary to the target sequence binds to the Cas enzymes Cas12 or Cas13, which then cleave at the desired target site. An additional interesting feature of Cas12 and Cas13 enzymes is that they show trans or collateral cutting activity. It means that on finding the target, the cleavage activity of Cas12 and Cas13 enzymes is not just restricted to the target DNA or RNA, but they can also cut any single-stranded non-targeted nucleic acid molecules in the vicinity. The collateral cutting activity of these enzymes has attracted scientists to build reporter systems in CRISPR diagnostics including the diagnostic test to detect SARS-CoV-2 which causes COVID-19.
Undoubtedly, CRISPR has changed the face of modern research and what has been achieved so far could just be the tip of the iceberg.
