CRISPR-Cas9

Introduction.

Life was miserable for Victoria Gray, a 34-year-old mother from the United States, due to complications of Sickle cell disease. Much of her life was punctuated by trips to the emergency room due to pain with life and career ambitions interrupted.[1]

In 2019, when Victoria Gray was exploring the possibility of getting a bone marrow transplant, her doctors suggested something different to treat her condition. She jumped at the chance and became the first patient with a genetic disease to get treated with a revolutionary gene editing technique using CRISPR–Cas9.[1]

Briefly, we will explore the history of this trending technique in the scientific community, its popularity, and associated benefits and risks.

What is CRISPR–Cas9?

This acronym means Clustered Regularly Interspace Short Palindromic Repeats and CRISPR-associated protein 9. Scientists use it as a genomic editing tool to cut a Deoxyribonucleic acid (DNA) by adding, removing, or altering sections of the sequence and triggering targeted changes to the genomes. It has two key molecules: Cas9 Protein and guide Ribonucleic acid (RNA).[2]

Genomic editing (gene editing), on the other hand, is a group of technologies that allow scientists to change an organism’s DNA. [3] Other tools include Zinc fingers nuclease (ZFN) and Transcription activator-like effector nuclease (TALEN).

How does it work?

The CRISPR – Cas9 system consists of two key molecules introducing a change (mutation) into the DNA. These are:

Cas9 is an enzyme that acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can be added or removed.[4]

RNA is also called guide RNA (gRNA). It consists of a small piece of pre-designed RNA sequence (about 20 bases long) like a script (a set of letters) located within a more extended RNA scaffold. The scaffold binds to DNA, and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome, ensuring the Cas9 enzyme cuts at the right point.[4]

This gRNA is designed to find and bind to a specific sequence in the DNA. It has RNA bases that are complementary to those of the target DNA sequence in the genome. The Cas9 follows the gRNA to the exact location in the DNA sequence and cuts across both strands of the DNA.[4]

The cell recognizes and tries to repair the DNA damage at this stage.

Where it all started?

The system evolved in microbes (Bacteria) because they needed to protect themselves from the viruses that attacked them. This protein is programmed to find and destroy the viral DNA in bacteria.[1]

The CRISPRs are regions in the bacterial genome that help to defend against invading viruses. These regions comprise short DNA repeats (black diamonds) and spacers (coloured boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated into existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules, which associate with and guide bacterial molecular machinery to a matching target sequence in the invading viral genome.[5]

History of CRISPR – Cas9.

In 1987, Japanese scientist Yohizumi Ishino and his team discovered a series of repeated sequences of unknown origin (Palindromes) that flanked other DNA sequences (spacers) in the bacteria Escherichia coli (E. coli).[2]

In 1993, a Dutch scientist, J.D Embden and his group discovered these spacer sequences between DNA repeats in Mycobacterium tuberculosis, the bacteria that cause tuberculosis.

However, in early 2000, Spanish scientist Francisco Mojica and Dutch Scientist Ruud Jansen became the first to refer to these discoveries as CRISPRs. They discovered that the sections of the DNA between the palindromes were coming from viruses. They described it as a molecular vaccination card for the bacteria, which helps them store pieces of DNA from the virus that have affected them over time. [2,6,7]

It was not until 2012 that a United States scientist, Jennifer Doudna and her counterpart Emmanuelle Charpentier took the discovery a step further by proposing that designing gRNA to target a specific region in the genome (i.e., reprogramming the CRISPR mechanism), the CRISPR system can be a “cut-and-paste” tool to modify genomes. [2,7]

Despite the considerable success recorded, scientists still worked hard to see that the system works effectively in humans. These efforts led to the discoveries made by Feng Zhan, a renowned Professor and George Church, in 2013.

They became the first to successfully adapt CRISPR-Cas9 for genome editing in human and mouse cells. In the same year, a University of California researcher, Jonathan Weissman, and his team invented CRISPR-dCas9 to create two variants, CRISPRa (for activation) and CRISPRi (for interference), which dial up or down gene expression at target genes. [6,7]

Reason for the Trend.

This system generated much excitement in the scientific community because of its versatility in its applications more than other editing tools. Some of the reasons include but not limited to:

1. Could correct genetic errors that cause disease.

In the summer of 2017, Scientists at Oregon Health and Science University used this system to delete one of the defective genes of a condition that causes stiffening heart tissue (Hypertrophic Cardiomyopathy). The outcome was impressive.[8]

2. It can eliminate the microbes that cause disease.

In 2017, a team of Chinese researchers successfully increased the resistance to HIV in mice by replicating a gene mutation that effectively prevents the virus from entering cells.[8]

3. Could create new, healthier foods.

Scientists from Cold Spring Harbour Laboratory in New York used the tool to increase the yield of tomato plants by editing the genes that determine tomato size, branching architecture and shape for a greater harvest.

The lead researcher, Professor Zachary Lippman, said, “Each trait can now be controlled in the way a dimmer switch controls a light bulb. We can now work with the native DNA and enhance what nature has provided, which we believe can help break yield barriers.”[8]

4. Could eradicate the planet’s most dangerous pest.

At the Imperial College London in 2016, researchers used this tool to target female reproduction of the mosquito that carries malaria through a gene drive system that influenced female-sterility traits into being likely to be inherited.

Also, it is becoming a go-to tool for drug discovery in biotech and pharma companies.[8]

How could CRISPR Cas-9 be introduced into Humans?

This novel technology could be either Ex-vivo or In-vivo.

Ex-vivo means “Outside the body.” Scientists introduce the gene editing molecules into the cells earlier taken out of a patient. The cells are edited and then injected back into the body; this is currently applied in treating SCD, just like in the case of Victoria Gray. There are ongoing clinical trials on this system, e.g., CART-T cells.[2]

In-vivo means “Inside the body”. The molecules are delivered into the human body using a virus as the vector, which holds it into the tissues where the editing is needed. This is harder, but scientists also work hard to actualize this technique. If success is recorded, it will be the best and most potent way of gene editing.[2]

Pros and Cons of the System.

The Pros.

Simple to Amend Target Region – Though setting up the system for the first time is not that simple, once the protocol is up and running, it is simple to ‘chop’ and change the setup to target alternative genomic regions for editing.[9]

Lots of Publications about the System – Since the first papers were published using CRISPR-Cas9, the number of publications on the technology has rocketed: more than 23,000 articles are listed on PubMed alone. This means thousands of labs, projects, and scientists worldwide use this system.

In a nutshell, the system is faster, cheaper, more adaptable, accurate, and excellent than other editing methods.[9]

The Cons.

Considerable Time Investment to Set Up from Scratch – Not all labs have an established genome-editing pipeline.

Not Always Efficient – Many factors can influence editing efficiency and severely affect the experiment. e.g., gRNA design

Off–Target Effects - In theory, the CRISPR-Cas9 system is precise, but in practice, it is not. It can create mutations elsewhere in the genome, known as ‘off-target’ modifications.[9]

Conclusion.

CRISPR-Cas9 has taken the world by storm. As one of the most significant discoveries of the twenty-first century, It has revolutionized clinical research by simplifying the study of diseases and accelerating drug discovery while profoundly influencing the development of crops, food, and industrial processes.

Finally, technological and ethical hurdles still stand between us and a future in which we feed the planet with engineered food, eliminate genetic disorders, or bring extinct species back to life.[10]

References.

1. Real Science. (2020, August 22) How Gene Editing is curing Disease [Video]. YouTube. https://www.youtube.com/watch?v=ezfwqmKC9Uc

2. World Science Festival. (2019, October 25) CRISPR in context: The New world of Human Genetic Engineering [Video]. YouTube. https://www.youtube.com/ watch?v=RNRZchHaKgw

3. MedlinePlus. (n.d) What are genome editing and CRISPR-Cas9? https://medlineplus.gov/ genetics/understanding/genomicresearch/genomeediting/

4. YgTopics. (n.d) What is CRISPR-Cas9? https://www.yourgenome.org/facts/ what-is-crispr-cas9

5. Pak, E. (n.d) CRISPR: A genome-changing genetic engineering technique. SITN . https:// sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/

6. Broad Institute. (n.d) CRISPR TIMELINE. https://www.broadinstitute.org/what- broad/areas-focus/project-spotlight/crispr-timeline

7. Campbell, M. (2019, October 14). Francis Mojica: The Modest Microbiologist Who Discovered and Named CRISPR. Genomics Research. https://www.technologynetworks.com /genomics/articles/francis-mojica-the-modest-microbiologist-who-discovered-and-named-crispr-325093

8. Tangermann, V. (n.d). A CRISPR FUTURE: Five Ways Gene Editing Will Transform Our World. Futurism. https://futurism.com/crispr-genetic-engineering-change-world

9. BitesizeBio. (2021, April 20). CRISPR-Cas9 Genome Editing: Weighing the Pros and Cons. https://bitesizebio.com/44187/crispr-cas9-genome-editing-system-weighing-the-pros-and-cons/

10. Fernandez, C. R. (2020, September 7) CRISPR-Cas9: The Gene Editing Tool Changing the World. LABIOTECH.eu. https://www.labiotech.eu/in-depth/crispr-cas9-review-gene-editing-tool/