If you have ever wondered what special device could edit genes in DNA and therefore correct errors in organisms, then advance to read more about this topic.
A genome editor called CRISPR-Cas9 is a unique biotechnology tool made up of a group of different technologies that allows scientists to target genes in DNA and edit it. This system first began introduction in 2012, and it allows for specific sights on DNA to be edited.
I was first introduced to gene editing technologies and CRISPR-Cas9 in my Anatomy and Physiology 12 class. In groups we got an overview of what CRISPR-Cas9’s role is in organisms from using paper cut-outs and even digital simulations. However, aside from these modelling activities I still had some questions about these devices, so I decided to investigate them more. Below, I have explained more about CRISPR-Cas9 while using credible online sources:
CRISPR-Cas9 is just one type of approaches to gene editing that has been quite recently developed and modified. This specific genome stands for Clustered Regularly Interspaced Short Palindromic Repeats with the associated protein, Cas9. The CRISPR part of CRISPR-Cas9 represents the specific type of technology that modifies the genes of living organisms. This editing device is based on the natural defense mechanism of bacteria and archaea. To explain this concept in more depth, there is a passage that explains it below. The ending Cas9 of CRISPR-Cas9 represents the protein that has a nuclease enzyme which cuts and unwinds DNA’s double helix. The gene editing technology, CRISPR can also have different types of proteins paired with it. Some of these different proteins can look like, CRISPR-Cas12 and Cas10. These different CRISPR-Cas systems are put into groups that represent type I, II, or III. For example, Cas3 is in type I systems and Cas 9 is in type II systems. All these different Cas’s are different proteins. The combination of CRISPR-Cas9 allows for a complex machine that collectively edits DNA.
The First Discoveries of CRISPR:
Crispr was first discovered in genes of DNA from Escherichia coli bacteria in 1987 by Ishina et al. from Osaka University in Japan. It was noticed that there was a naturally occurring genome editing system that bacteria used as an immune response. When bacteria are infected with viruses, some of the virus’s DNA can be inserted into their own DNA. This allows for the bacteria’s DNA to create segments known as CRISPR. Then the bacteria produce RNA segments from CRISPR that recognizes and attaches to the particular region of the virus’s DNA. Then the Cas9 protein will cut the virus’s DNA which will then disable the virus. Also, the CRISPR system allows bacteria to remember the virus if it attacks again. From these first discoveries, CRISPR-Cas9 could be discovered and researched by other scientists as well. For example, for the first time in history a Nobel prize was awarded to two women, Emmanuelle Charpentier and Jennifer Doudna which made the key discoveries in terms of DNA manipulation with the CRISPR-Cas9 system.
As stated before, the structure of CRISPR-Cas9 consists of the joining of the Cas9 protein which contains nuclease enzymes that play an important role in cutting DNA and therefore altering it. These proteins are usually extracted from specific bacteria like Streptococcus pyogenes which usually causes most of the strep infections in people. some other Cas proteins are also extracted from the largest known bacteria-infecting viruses, called bacteriophages.
Cas9 protein
CRISPR-Cas9
When you get injected with the CRISPR-Cas9 treatment, the cells in the treatment produce the CRISPR and have the Cas9 proteins joined to it as well. However, for the gene technology to work it needs to know where in the body it needs to go, or more specifically it needs to know what gene on DNA it needs to target. Researchers discovered how CRISPR-Cas9 could edit a living organism DNA. Scientists first created a small piece of guide RNA that contained a sequence of about 20 nucleotides long, with complementary base pairings consisting of adenine pairing with uracil and cytosine pairing with guanine (nitrogenous containing bases from nucleotides). This gRNA would bind to the DNA segment that needs to be cut and altered. This gRNA also guides CRISPR-Cas9 to the sequence of DNA that is targeted for the editing and cutting.
Guiding RNA & DNA Segment
When the guide RNA is added to Cas9, it will guide it to the target sequence on DNA. This target sequence can be about any sequence long, but it has to occur near a PAM motif (PAM – Protospacer Adjacent Motif). PAM helps CRISPR-Cas9 find the beginning of a target sequence so that the proteins cuts are accurate. When Cas9 is guided to the right location it will recognize and bind to PAM, which would be a three-nucleotide sequence motif. This motif can consist of any nucleotide first followed by two guanines, but this order will need to be looked at a DNA sequence in a 5’ to 3’ direction (for reference: 5’-N-G-G-3’). Also, this sequence motif occurs all throughout the human genome.
PAM sight location
Once Cas9 binds to the PAM motif, it starts unwinding or ‘cutting’ the DNA double helix moving upwards away from PAM. During this point and location, if the DNA perfectly matches a sequence of about 20 nucleotides within the guide RNA, the DNA and matching RNA will then bind through complementary base pairing. However, if the sequence of the unpaired DNA strand is not an exact match to the 20-nucleotide sequence in the guide RNA, Cas9 then detaches from DNA. In this situation, Cas9 will keep binding to other PAM sequences until it finds the matching target DNA. The DNA will then zips back up into its double helix if there isn’t an exact match.
Once the DNA and RNA complementary base pair with one another, this allows for Cas9 to change its three-dimensional structure and activates its nuclease activity. This activity is the action of Cas9 cleaving, or cutting both DNA strands, going three nucleotides up from PAM (towards the 5’end). These cuts are very specific in DNA, and they include two cleavage sights where DNA’s two strands will be cut.
Aftermath of Cas9 Cleaving both DNA Strands
When the two strands of DNA are cut, the cell will attempt to repair the break. Or more specifically, the cells enzymes will want to repair these breaks. This repair process naturally results in errors and will lead to mutations that may inactivate a gene. Inactivating a gene means it will not be able to be expressed as a protein.
There are two different repair mechanisms that CRISPR-Cas9 uses which are called nonhomologous end joining (NHEJ) or homology-directed repair (HDR).
NHEJ is the most frequent used and faster repair mechanism. This method is faster as it does not use a template to join the broken DNA ends together. However, this repair mechanism is sometimes error-prone that can cause mutations in the target DNA sequence. Still, errors are rare and if the break is repaired correctly during this process, Cas9 will recognize the target sequence once again and cleave it. As the Cas9 keeps on repeating the cycle of cleavage and repairs, it will eventually result in a mutation.
This type of mutation will be random, and it will happen between the DNA target sequence. If the target sequence is beside a gene’s coding region, the mutation will possibly inactivate that gene.
NHEJ Repair Mechanism
The second repair mechanism is HDR, which is a less error-prone process than NHEJ to repair the DNA break. However, HDR uses a template to accurately repair the break on DNA which is usually from a homologous chromosome. Scientists may provide an extra DNA repair template and Cas9-guide RNA complex. From this extra addition, the cell’s repair system will be “tricked” into using the repair template to fix the break by using HDR. From designing different repair templates, scientists can also change the target DNA sequence into a whole new sequence. Doing this can also correct an existing mutation by replacing it with a nonmutated sequence of DNA.
HDR Repair Mechanism
Further Questions:
How can CRISPR-Cas9 be used to our benefit?
Since CRISPR-Cas9 allows genetic material to be added, removed, or altered at particular locations in genes it can make some very important changes in an organism’s genetic code. It allows us to manipulate genomes of organisms in scientific experiments to allow for new discoveries and breakthroughs. Using CRISPR-Cas9 may also allow us to modify certain characteristics of important crops and animals. Or it also has the capability to introduce revolutionary changes in medicine, for example, allowing more treatments of genetic diseases or viruses like COVID-19.
Also, CRISPR-Cas9 can be injected directly into the patient which in most other gene editing is not the case. With CRISPR-Cas9 having RNA involved in its system, it can be more efficient and easily modified with targeting of multiple sights at a time compared to other gene editing approaches. Finally, CRISPR-Cas9 is also faster, cheaper, more accurate, and more efficient than other gene editing tools.
During class what models did you used to learn?
As I stated before, during class we learned about CRISPR-Cas9 with two different modelling activities to learn about a complex microscopic process. We first used paper cut-outs and a guide to help us while going through CASPR-Cas9’s roles. After using the paper cut-outs, we used a digital simulation which also provided information on what was happening on our screens.
In what ways did the model accurately reflect the process? In what ways did the model misrepresent the process? What changes could be made to the activities to make them better represent the actual process?
The paper cut out modelling activity was more hands on and allowed for us to see each role of CRISPR-Cas9 clearly. I felt that I could get a thorough understanding of CRISPR-Cas9 especially with working with the different cut out components in front of me and having to see what was truly going on. However, this model didn’t accurately represent the structure and configuration of all the components of the CRISPR-Cas9 process. For example, it represented RNA and Cas9 as constant structures when in the digital model they looked more intricate.
The digital simulation diffidently provided a more accurate image of what CRISPR-Cas9 would look like in a living organism. However, even though there was instructions and lots of information given it wasn’t as easy to understand as doing the hands on paper activity. In seemed like the process had a lot less steps than when I used the paper model to see CRISPR-Cas9’s roles. Some changes that could be made to the paper model to better represent the actual process is to make the different components in the process of gene editing more realistic. This will allow for a better understanding of the whole process. Also, the digital simulation could benefit with having more steps and going into more depth about the different components and roles of gene editing done by CRISPR-Cas9.
Models are commonly used to communicate scientific concepts to non-scientific audiences. Do you think this is an effective way to educate students and/or the public about science?
I think using models to communicate scientific concepts to non-scientific audiences is an effective way to educate students and/or the public about science. Models allow for ideas and explanations to be represented much more easily. They also provide options for individuals who need extra support in understanding a concept. Models can help describe, understand, and predict processes occurring in the world. Individuals who are learn better with visual references may find it easier to grasp a concept as well. They also allow for learners to be more independent in their learning since they are the one having to figure out what is going on in front of them, which may facilitate critical thinking.
Word bank:
Genome – the genome is the entire set of DNA instructions found in a cell.
Genes – Genes are segments of DNA.
Archaea – Archaea are a group of micro-organisms that are similar to bacteria.
RNA – A molecule that can read the genetic information in DNA.
Cas9 – Cas9 is a nuclease, a type of enzyme that cuts DNA.
Enzyme – Enzymes are proteins that help speed up chemical reactions in our bodies.
Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR is short sequences of DNA that are the read the same in both directions (5’ to 3’) and are in between segments called spacers.
Fun Facts:
The gene editing technology CRISPR has been very fascinating so far. Below is a list of some further discoveries while using CRISPR:
- Turning animals into organ donors
- Creating new treatment for cancer and blood disorders
- Eliminating mosquitos
- Modifying human embryos
- Making new and improved fruit
Further Questions:
- Will gene editing technologies like CRISPR-Cas9 be the new therapy used to get rid of diseases?
- Is CRISPR-Cas9 just the start of many different revolutionary medical advancements?
- Will gene editing be able to modify living organisms in un-humane ways?
- Could individuals be born without any diseases or health issues if embryos can be modified?
Sources:
BioInteractive, hhmi. (2012). CRISPR-Cas9. https://media.hhmi.org/biointeractive/click/CRISPR/
Medline Plus, N. L. of M. (2022). What are genome editing and CRISPR-Cas9?: Medlineplus Genetics. MedlinePlus. https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/
Lab, Z. (2014, December 17). Questions and answers about CRISPR. Broad Institute. https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr
Saey, T. H. (2020, October 9). Explainer: How CRISPR works. Science News Explores. https://www.snexplores.org/article/explainer-how-crispr-works/
Mullin, E. (2020, May 18). The 7 craziest ways CRISPR is being used right now. Medium. https://onezero.medium.com/the-7-craziest-ways-crispr-is-being-used-right-now/
Gostimskaya, I. (2022, August). CRISPR-Cas9: A history of its discovery and ethical considerations of its use in genome editing. Biochemistry. Biokhimiia. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9377665/
PAM digital image – Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014;513(7519):569-573.