CRISPR-Cas9: A Beginner's Guide!

Written in June 2024

Hello, future scientists! Welcome to "Science Unveiled," your go-to blog for making science fun and easy to understand. Today, we will dive into CRISPR-Cas9. Imagine a world where we can edit the DNA of living organisms to correct genetic disorders, enhance crops, and even eradicate diseases... Sounds too good to be true? Many people see CRISPR-Cas9 as a complex and daunting system reserved for experts... While it is indeed sophisticated, by the end of this blog, you'll understand the basics of how it works and why it is so exciting!!

As you read through this blog, you will come across the following sections. Enjoy!

The CRISPR System Broken Down!

Understand a very simplified and easy-to-comprehend breakdown of the CRISPR system (found in bacteria!)... Learn about its discovery and development, the basics of its key components, and its advantages and disadvantages.

Deep Dive! 🥽

Delve deeper into complexities! Gain insights into advanced mechanisms, ongoing challenges, and the more in-depth phases of the CRISPR-Cas system.

What do you think?

Engage in thought-provoking polls, discussions, and case studies. It is a lot of fun!

References

See the sources and scholarly works consulted for information and citations throughout this article.

The CRISPR System Broken Down!

What is the CRISPR System?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. These are specific sequences found in the DNA of bacteria and archaea that serve as a part of their immune system.

Imagine CRISPR as a 'genetic memory book' for these microorganisms. Within this 'book' are sections called CRISPR arrays. These arrays are made up of repeating DNA sequences with unique parts in between called "spacers." Each spacer is just a unique sequence derived from the viral DNA. You can think of spacers as a 'snapshot' of a past virus the bacterium has faced.

When a virus attacks, the bacterium captures a piece of the virus's DNA and adds it to its CRISPR array. This process is akin to writing a new memory in the 'book'. If the same virus tries to attack again, the bacterium uses these memory snapshots to recognize and combat the invader. The CRISPR system does this by transcribing a particular RNA called pre-crRNA (pre-CRISPR RNA) —which is subsequently processed into smaller CRISPR RNAs— that acts like a guide. This guide directs particular proteins called Cas proteins to the matching viral DNA sequence, using the spacer as a template.

The History of CRISPR-Cas9

The journey of CRISPR began in 1987 when Yoshizumi Ishino and his team at Osaka University discovered unusual DNA sequences in—you guessed it—E. coli (surprise, surprise 🙄). This set of unusual DNA sequences was later named CRISPR by Francisco Mojica. Mojica hypothesized that these sequences played a role in microbial immunity by storing viral DNA fragments, which was then validated in 2007. However, the real breakthrough came in 2012 when Jennifer Doudna and Emmanuelle Charpentier demonstrated how the CRISPR system could be repurposed for programmable gene editing. Their work simplified the new CRISPR-Cas system to two key components: the Cas nuclease and guide RNA (gRNA).

Key Components: Cas Proteins and Guide RNA

CRISPR-Cas9 relies on two main components: Cas proteins and guide RNA (gRNA).

Firstly, let us discuss Cas proteins. Cas proteins are basically just a diverse family of enzymes that recognize, bind to, and cleave (cut) nucleic acids—either DNA or RNA— and are guided by gRNA.

Next, the gRNA is a synthetic RNA molecule that directs the Cas9 protein to its specific target sequence in the genome. It is composed of two parts: the CRISPR RNA (crRNA) that binds to the complementary DNA sequence and the trans-activating CRISPR RNA (tracrRNA) that stabilizes the complex, allowing Cas9 to locate and bind to the target site. This precise interaction between Cas9 and gRNA enables the high accuracy of CRISPR-Cas9 technology.

Read the deep dive to gain a more in-depth understanding of the structure and function of these Cas proteins and gRNA.

Advantages and Disadvantages

CRISPR-Cas9 offers numerous advantages, but it also comes with challenges.

On the pro side, CRISPR-Cas9 allows for highly specific targeting of genes, enabling precise edits at the molecular level. This precision is one of its greatest strengths, especially when compared to other CRISPR systems that use different Cas proteins (due to factors such as the ability to customize the gRNA with great accuracy, the reliance on PAM sequences —which are covered in the deep dive section—, the gRNA's dual structure, etc)... Additionally, the technology is highly versatile, being applicable across various organisms and cell types, making it a powerful tool for both research and therapeutic applications. Furthermore, CRISPR-Cas9 is relatively cost-effective (well... not exactly "cheap"), but it's definitely more affordable compared to earlier gene-editing methods like Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), making it more accessible to a broader range of scientists and institutions.

However, there are also significant drawbacks to consider. One challenge (explained in the deep dive section) is that CRISPR-Cas9 relies on creating double-stranded breaks in the DNA, which sometimes can lead to off-target effects. These unintended breaks can cause harmful mutations, making the genome unstable which can introduce errors, such as small insertions or deletions, or even larger disruptions like DNA rearrangements. These errors can disrupt important genes, cause cellular stress, and may even lead to cell death or halt cell growth. Another important consideration is that delivering CRISPR components to the right cells in a living organism is still a significant challenge. While CRISPR-Cas9 is highly precise once inside the cell, ensuring that it reaches the intended cells effectively remains a major obstacle in developing practical therapeutic applications.

Deep Dive! 🥽

Cas Proteins and Guide RNA

To truly appreciate how CRISPR-Cas9 functions, we need to take a closer look at the proper structures and functions of the key components of this technology.

1. Cas Proteins (Cas9 Specific)

As you know, Cas proteins are basically complex enzymes. These complex enzymes comprise of several functional domains that each play a critical role. Considering that Cas9 is the most widely used Cas protein in gene editing, let us take Cas9 as an example.

Cas9 contains two key nuclease domains: RuvC and HNH. The RuvC domain is responsible for cleaving the non-target strand of DNA. And the HNH domain cleaves the target strand. Imagine DNA as a ladder with two sides; the RuvC domain cuts one of those sides, while the HNH domain works on the opposite side. So, together, these nuclease domains introduce a double-strand break at the target site, which is a crucial step in the gene-editing process. This double-strand break will result in the cell trying to repair it. Depending on how the repair happens, this can either disrupt the gene (which could potentially cause the loss of function, genetic diseases, other unintended effects, etc... which raises concerns) or allow for new genetic material to be added if a template is provided.

Another vital component of Cas9 is its RNA-binding domains, which interact specifically with the gRNA to ensure that the gRNA is correctly aligned with the Cas9 protein to accurately recognize the target DNA sequence.

One more critical domain Cas9 has, which we should note, is specifically designed to recognize the Protospacer Adjacent Motif (PAM), a short DNA sequence immediately following the target DNA sequence. This domain is essential as the Cas9 protein will only bind to DNA sequences adjacent to a PAM, adding an extra layer of specificity to the targeting process.

2. Guide RNA (gRNA)

The gRNA is composed of two main parts: CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).

The crRNA binds to the complementary DNA sequence of the target gene. It contains the 20-nucleotide sequence called the 'spacer' (which you should be familiar with now), that is complementary to the target DNA sequence. On the other hand, the tracrRNA is essential for forming a complex with the Cas9 protein and for the proper maturation of crRNA from pre-crRNA.

It is important to note that while in the natural CRISPR system, crRNA and tracrRNA are separate molecules, in the engineered CRISPR-Cas9 system used for gene editing, these two RNA molecules are often fused into a single guide RNA (sgRNA). This fusion simplifies the process and improves efficiency. The sgRNA is the one that guides the Cas9 protein to the specific DNA site where the double-strand break will be introduced.

Once the gRNA is introduced into the cell, it forms a complex with the Cas9 protein which then searches the genome for a sequence that matches the 20-nucleotide spacer region of the gRNA. Upon finding a match, the Cas9 protein undergoes a conformational change that allows it to recognize the PAM sequence adjacent to the target DNA, which is crucial to successfully bind and cut the DNA.

Why Cas9?

Let me ask you a question: Which do you hear more often— CRISPR-Cas9, CRISPR-Cas12, or CRISPR-Cas13?

Exactly! You hear about CRISPR-Cas9 the most! But why is that? Let me explain...

1. Simplicity

Compared to other Cas nucleases, Cas9 is relatively straightforward to use as it only requires a single guide RNA (sgRNA) to direct it to the target DNA sequence. This makes designing and implementing simpler.

2. PAM Sequence Requirement

As you know, Cas9 requires a specific Protospacer Adjacent Motif (PAM) sequence adjacent to the target DNA sequence to bind and cut effectively. This requirement helps ensure precise targeting and reduces the chances of off-target effects in comparison to other CRISPR nucleases that may have less stringent PAM requirements (less common, more specific, etc)

3. Versatility

Cas9 can be engineered to target a wide range of DNA sequences. This can be done by just simply altering the gRNA. This allows for various applications in gene editing.

4. Performance

Cas9's robust performance and ability to properly create double-strand breaks (DSBs) in DNA has made it a go-to choice for many gene editing opportunities. It can induce DSBs effectively and facilitate subsequent repair processes which can introduce or correct DNA sequences.

The Phases of The CRISPR-Cas System (HUMAN Gene Editing)

The CRISPR-Cas gene editing system operates with the help of three main stages.

1. Adaptation (Space Acquisition)

In the context of human gene editing the terms 'adaptation' or 'acquisition' don't really apply the same way it would for bacteria. Instead, scientists design a synthetic guide RNA (gRNA) to match the specific DNA sequence they want to edit in the human genome. This guide RNA sort of acts like a GPS, as mentioned before, directing the CRISPR-Cas9 system to the exact location in the DNA where changes are desired.

This step involves the careful design and preparation of the gRNA, which is crucial for ensuring that the system targets the right part of the genome.

2. crRNA Biogenesis

Once the gRNA is introduced into human cells, it pairs with the Cas9 protein to form a complex that is ready to perform the gene-editing task. The gRNA is engineered to contain two components: the CRISPR RNA (crRNA) —which binds to the specific target sequence in the DNA— and the trans-activating CRISPR RNA (tracrRNA) —which stabilizes the complex and helps guide Cas9 to the right spot in the genome.

This stage involves the assembling of the components meant to find and interact with the target DNA sequence in cells.

3. Target Interference

The final stage is where the actual gene editing occurs. The gRNA-Cas9 complex travels through the cell, searching for the specific DNA sequence that matches the guide RNA. Once the target DNA is found, Cas9 makes a precise cut at that location in the genome. In human gene editing, this cut can be used in two main ways.

(i) Gene Disruption: If the goal is to disable a gene, the cell’s natural repair processes will attempt to fix the cut, often introducing small errors that disrupt the gene's function.

(ii) Gene Insertion/Correction: If scientists want to add a gene or correct a gene, they can provide a DNA template along with the CRISPR-Cas9 system. The cell uses this template to repair the cut, incorporating the new or corrected genetic information into the genome.

Ethical Considerations

Editing human DNA raises profound questions. One major concern is the potential for increased inequality, as access to gene-editing technology might be restricted to those with financial means. This could lead to a society where genetic “enhancements” are only available to the wealthy. Those who can afford gene-editing technologies might gain advantages such as enhanced intelligence, physical abilities, or other traits that result in increased social and economic disparities. Over time, this could worsen existing inequalities and create a genetic divide, exactly like those science fiction scenarios where only some have special abilities and the rest are left behind... (it's a dystopian idea that, while once only imagined in fantasy books, might become a real possibility if we’re not careful.)

Another one of the many concerns is the possibility of creating “designer babies” which refers to children whose genetic traits are selected or altered for non-medical reasons, poses ethical dilemmas about human diversity and the loss of genetic variability. It poses the risks of inequality and parental pressure as well. Designer babies are a scary possibility that can challenge our understanding of what constitutes a "normal" or "acceptable" human condition.

As we advance with CRISPR-Cas9, it is crucial to develop robust ethical guidelines and engage in global discussions to ensure the technology is used responsibly and equitably.

What Do You Think?

Polls and Surveys

Comment Why Down Below!!

Which area/field of research should prioritize the use of CRISPR-Cas9 technology?

• Genetic Disorders (e.g., cystic fibrosis, sickle cell anemia
• Agriculture (e.g., crop enhancement, pest resistance)
• Environmental Conservation (e.g., gene drives for invasive s
• Other (please specify in comments!)

Do you believe CRISPR-Cas9 should be used to edit the human genome for non-medical reasons (e.g., enhancing physical or intellectual traits)?

• Yes
• No

How concerned are you about the potential for CRISPR-Cas9 to create social inequality?

• Very Concerned
• Somewhat Concerned
• Not Concerned

Discussion Questions

1. Is it ethical to use CRISPR-Cas9 to alter human embryos, knowing that these changes will be passed on to future generations?

            2. Should access to gene-editing technologies like CRISPR-Cas9 be regulated to prevent their use for non-medical enhancements?

            3. What responsibility do scientists and governments have in ensuring that CRISPR-Cas9 is used safely and ethically?

Case Studies!

DISCLAIMER: The case studies presented in this blog are entirely fictional and are created for educational purposes only! They are not based on real individuals, events, or organizations. Any resemblance to actual persons —living or dead— or actual events is purely coincidental.

CASE STUDY 1 🧠

In 2027, Emily and Jack Thompson, a couple living in San Francisco, learn that they are both carriers of a recessive gene mutation that causes spinal muscular atrophy (SMA), a severe genetic disorder that leads to muscle wasting and early death. The Thompsons desperately want to have children but are concerned about passing on this devastating condition. Their genetic counselor suggests using CRISPR-Cas9 to edit the embryos during in vitro fertilization (IVF) to ensure their future child does not inherit SMA.

• If they decide to move forward, who should bear the cost of the procedure... should it be covered by their health insurance, or should it remain an elective, out-of-pocket expense?

  
  

• Should Emily and Jack proceed with CRISPR-Cas9 gene editing to eliminate the risk of SMA, considering the long-term implications for their child and future generations?

  
  

• Should there be a mandatory waiting period for parents considering this procedure to ensure they have fully considered the ethical implications?

CASE STUDY 2 🧠

In 2031, a luxury biotech firm called Genetica, based in Luxembourg, begins offering a premium service that allows wealthy clients to select specific traits for their unborn children. The Johnsons, a prominent and affluent couple working in real estate, decide to use this service to enhance their future child's intelligence, physical appearance, and athletic ability. They pay $450,000 for the procedure, which is conducted in a state-of-the-art lab. The child, Emma Johnson, is born in January 2029, with enhanced traits as specified by her parents.

• Should Emma be informed about this, and at what age?

  • If she does find out, how might her experience of growing up differ from that of other children, knowing that she was genetically modified for specific traits?

    
    

• Could this widespread use of such genetic modifications by only the wealthy lead to a new form of social inequality or discrimination?

  
  

• Should there be legal restrictions on which traits can be modified using CRISPR-Cas9, or should parents have the freedom to choose any traits they desire for their children?

CASE STUDY 3 🧠

In 2029, Dr. Saran Ramirez, a leading geneticist at the World Health Organization (WHO), proposes a groundbreaking project to combat malaria in sub-Saharan Africa. Her team plans to use CRISPR-Cas9 to genetically modify the mosquito species Anopheles gambiae to render them incapable of transmitting the malaria parasite. The project, set to begin in March 2031, is expected to spread the modified genes rapidly through the mosquito population within five years. However, there are concerns about the potential ecological impact and the ethical considerations of releasing genetically modified organisms into the wild.

• What specific ecological risks should Dr. Ramirez and her team assess before releasing the genetically modified mosquitoes, and how can they mitigate these risks?

  • Should they conduct a smaller-scale trial before a full release?

    
    

• How should local communities in sub-Saharan Africa be involved in the decision-making process for this project?

  • Should they have the right to veto the release if they feel uncomfortable with the potential risks?

References

Anders, C., Niewoehner, O., Duerst, A., & Jinek, M. (2014). Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513(7519), 569–573. https://doi.org/10.1038/nature13579

Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science, 339(6121), 819–823. https://doi.org/10.1126/science.1231143

Doudna, J. A., & Charpentier, E. (2014). The New Frontier of Genome Engineering with CRISPR-Cas9. Science, 346(6213), 1258096–1258096. https://doi.org/10.1126/science.1258096

Hsu, Patrick D., Lander, Eric S., & Zhang, F. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262–1278. https://doi.org/10.1016/j.cell.2014.05.010

Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169(12), 5429–5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829

Kosicki, M., Tomberg, K., & Bradley, A. (2018). Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology, 36(765–771). https://doi.org/10.1038/nbt.4192

Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J., & Soria, E. (2005). Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. Journal of Molecular Evolution, 60(2), 174–182. https://doi.org/10.1007/s00239-004-0046-3

Nishimasu, H., Ran, F.  Ann, Hsu, Patrick D., Konermann, S., Shehata, Soraya I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001

Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013). One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 153(4), 910–918. https://doi.org/10.1016/j.cell.2013.04.025