CRISPR-Cas9: a tool that will revolutionise humanity

If you have stumbled across this article, you may be wondering, "CRISPR? What kind of name is that? How will this revolutionise humanity?" Well, be amazed as I go through its workings and how it holds immense promise to the future of biotechnology and genetic modification. From manipulating agricultural crops to increase nutritional values to combatting cancer, CRISPR's transformative power goes beyond boundaries.

What is CRISPR?

CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is a tool that was developed by two women, Emmanuelle Charpentier and Jennifer Doudna, and was awarded the Nobel Price in Chemistry in 2020. CRISPR was first discovered in the sequences of DNA from Escherichia coli (E. coli) in 1987 by Ishino et al. in Osaka University.

CRISPR can be taught of "genetic scissors". This tool enables geneticists and medical researchers to edit parts of the genome by removing, adding, or altering sections of the deoxyribonucleic acid (DNA) sequence. The genome is basically the organism's complete set of genetic instructions, and inside the genome? DNA sequences.

How does it work?

The CRISPR-Cas9 system consists of 2 key molecules that cause changes in the DNA. The enzyme, called Cas9, cuts the 2 strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. The other component, guide RNA (gRNA), is the "assistant". It consists of a small piece of pre-designed RNA sequence located within a longer RNA scaffold. The scaffold part binds to the DNA and the pre-designed sequence guides the Cas9 enzyme to the right part of the genome a.k.a. the target site.

The gRNA is designed to find and bind to a specific sequence in the DNA. The gRNA has bases that are complementary to the target DNA in the genome, so the gRNA can only bind to the target sequence and no other regions.

How was this developed?

This defense mechanism is seen in bacteria and archaea. When a bacterium is infected by a virus, it uses a Cas nuclease (similar to Cas9) to snip off a piece of viral DNA known as a protospacer. This fragment is stored in the bacterial genome with fragments from other viruses that have previously infected the cell, which is basically an immune memory (even we humans have a similar immune memory!). These viral fragments are placed between repeated palindromic sequences, giving CRISPR its unique name.

When the bacterium is again infected with the same virus, the bacteria can recognise it and destroy it with the Cas9 enzyme. The Cas9 activity relies on CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). The crRNA is complementary to the viral spacer that was stored after the original infection, while the tracrRNA serves as a scaffold. These two RNAs form the gRNA. Before cutting, the Cas9 acts as a search tool, checking the viral DNA for the protospacer adjacent motif (PAM), a short sequence downstream of the target site. When it recognizes PAM, Cas9 checks the region upstream. If it locates the target provided by the gRNA, it will create a double-stranded break (DSB).

When Doudna and Charpentier found out about this natural mechanism, they asked themselves whether this same enzyme can create desired cuts at an organism's genome if given a separate gRNA sequence.

The answer is yes.

Cellular DNA editing pathways: NHEJ and HDR

The natural gRNA complex can be engineered into a chimeric single guide RNA (sgRNA), which is a simple and cost-effective method of genetic manipulation. CRISPR-Cas9 gene editing works by creating DSBs in the DNA and then taking advantage of cellular DNA editing pathways. The common ones are non-homologous end joining (NHEJ) and homology-directed repair (HDR).

NHEJ is the predominant cellular repair pathway that mends DSBs in most eukaryotes. It works by rejoining the blunt ends of DNA back together with minor processing, which usually takes around 10 minutes and thus is referred to as a "quick fix" mechanism. NHEJ repairs the DSB by directly ligating the broken DNA ends together, without the need for a homologous DNA template. The process involves several steps:

1. Recognition and binding: Proteins involved in the NHEJ pathway recognize the broken DNA ends and bind to them.

2. Trimming: The broken DNA ends may contain damaged or mismatched nucleotides. Nucleases trim away these damaged portions to generate clean DNA ends for ligation.

3. Alignment and ligation: The DNA ends are aligned and joined together by DNA ligases, which catalyze the formation of phosphodiester bonds, connecting the DNA strands.

NHEJ can result in small insertions or deletions (indels) at the site of the repaired DSB. These indels can cause disruptions or alterations in the genetic sequence, leading to gene mutations.

HDR stands for homology-directed repair and is the second most common repair mechanism for DSBs in eukaryotes. Unlike NHEJ, HDR depends on a homologous repair template, such as a sister chromatid, to repair the broken DNA. HDR is thus active in the late S and G2 phases when sister chromatids are present in the cell. As a result, DNA is often repaired faithfully with no indel formation. The process of HDR includes:

1. DSB induction: A specific site in the DNA is targeted for cleavage, typically using techniques like CRISPR-Cas9. This creates a DSB at the desired location.

2. DNA end resection: The broken DNA ends are processed by nucleases to generate single-stranded DNA (ssDNA) overhangs. This resected DNA then serves as a primer for repair.

3. Template search and alignment: The resected ssDNA searches for a homologous sequence in the genome or an exogenous DNA fragment. Once a homologous sequence is found, the resected ssDNA aligns with the complementary sequence in the template.

4. DNA synthesis and repair: DNA polymerases extend the resected ssDNA using the homologous sequence in the template as a guide. This leads to the synthesis of a new DNA strand that complements the damaged DNA.

5. Ligation: The newly synthesized DNA strand is ligated with the intact DNA strand, sealing the repaired DNA molecule.

Is CRISPR ethical?

This technology holds much promise towards science and its advancement, but it has some bioethical caveats.

One of them is ecological imbalance. In studies using RNA-targeted gene editing methods based on CRISPR-Cas9, nontarget effects should be examined in depth. Gene drift is the change in frequency of an existing gene variant in the population due to random chance. Since gene drift will persist in a population, possible off-target mutations will continue in each generation. In addition to that, the number and effect of mutations may increase as generations progress. Another concern is the possibility that genes can be transferred to other species in the environment. Transferring the regulated sequences to other species may transmit negative characteristics to the associated organisms.

Another problem is using this technology in the human germline, i.e. cells that form eggs and sperm. In 2015, the editing of the human germline performed by Chinese scientist Huang and his team with CRISPR-Cas9 raised serious ethical and moral issues. Some of the ethical dilemmas of genome editing in the germline arise from the fact that changes in the genome can be transferred to the next generations. Therapeutic genome editing in somatic cells generally does not cause significant concerns, but the application of it in the germline is considered more problematic because of the risk of causing various mutations and side effects and transferring undesirable changes to future generations.

CRISPR in action

CRISPR has a plethora of uses in various industries and fields. CRISPR is becoming the common tool for drug discovery and development in biotech and pharma companies. In academic research labs, the gene-editing tool is being used to modify the genome of all sorts of organisms to study the function of any gene of interest. This technology can modify DNA with greater precision than existing technologies, gaining the edge over other mutagenic techniques like zinc-finger nucleases (ZFNs) or transcriptor activator-life effector nucleases (TALENs).

Another application of CRISPR is in the agricultural sector. It is used in rice to increase yields in order to meet the demand for our ever-growing population, heightened by its importance in multiple cuisines and the fact that it is overly susceptible to negative environmental factors such as climate change. Another trial is being carried out in the European Union (EU) in wheat to remove the gluten so that people with Celiac disease are able to consume it without life-threatening consequences.

One of the most important applications of CRISPR is in the field of oncology. CRISPR can cure a number of genetic diseases ranging from blood diseases like sickle cell anaemia to cancer. The data from clinical trials released recently has demonstrated that CRISPR therapy has been successful in treating patients with sickle cell anaemia and beta thalassemia.

In conclusion, this technology holds immense potential for the future of genetic modification. Varying industries are all unified by this gizmo for the greater good. However, it also raises ethical considerations as well. It is critical to approach its implementation with responsible behaviour in order to achieve scientific progress.

Citations

"What Is CRISPR: The Ultimate Guide To CRISPR Mechanisms, Applications, Methods & More." Synthego, https://www.synthego.com/learn/crispr. Accessed 26 May 2023

"CRISPR Editing is All About DNA Repair Mechanisms." Synthego, https://www.synthego.com/blog/crispr-dna-repair-pathways. Accessed 27 May 2023

"CRISPR in Agriculture: An Era of Food Revolution." Synthego, https://www.synthego.com/blog/crispr-agriculture-foods. Accessed 27 May 2023

"Diseases CRISPR Could Cure: Latest Updates On Research Studies And Human Trials." Synthego, https://www.synthego.com/blog/crispr-cure-diseases. Accessed 29 May 2023

Ayanoğlu, Fatma Betül, et al. “Bioethical Issues in Genome Editing by CRISPR-Cas9 Technology.” Turkish Journal of Biology = Turk Biyoloji Dergisi, U.S. National Library of Medicine, 2 Apr. 2020, www.ncbi.nlm.nih.gov/pmc/articles/PMC7129066/. Accessed 03 June 2023