Introduction:
Last week, we began our research about biotechnology techniques, we mainly focused on CRISPR-Cas9, a tool of biotechnology scientist have discovered. We got the chance to explore this process by building a paper model of “kick-in” and “kick-out” genes of CRISPR-Cas9. Then we deepened our understanding and how CRISPR works by visiting a 3D website model, that provided us with insightful descriptions in a step-by-step processes of CRISPR. This process gave us a clear insight about the process of DNA modifications. This tool have been long used for modifying, investigation, and studying DNA alterations among different organisms, including humans. It also, been a good tool for agricultural and medical development. As a significant component of biotechnology CRISPER-Cas9 system discovered in bacteria and first began in 2012. As this tool developed it became more effective for matter such as designer babies.
CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats” and -Cas9 stand for “CRISPR-associated protein 9.” CRISPR represents that repeating sequences of nucleotides in the DNA genome of bacteria. Cas9 is an enzyme that cuts the DNA strands and acts as a molecular scissors. It’s the major principle of gene-editing system derived from bacterial immune defense mechanisms. The CRISPR region contains short DNA repeats that, together with Cas9, help bacteria recognize and cut viral DNA. The structure of CRISPR-Cas9 involves of a guide RNA, Cas9 enzyme, and a DNA endonuclease enzyme. The process of DNA editing using CRISPR-Cas9 consisted of two particular molecules that are responsible for the process: a guide RNA which is 20-nucleotides long sequence that target a specific DNA sequence that is required to be modified by scientist, and the Cas9 endonuclease (nucleic acids). recognize and bind 3 nucleotides sequence called the PAM (Protospacer Adjacent Motif) which are found in large amount throughout the genome, and cleave at a specific gene location. This allows for precise genetic modifications. This process result in an isolated DNA strand that is either repaired or removed. Alternatively, if it wasn’t complimentary to the (gRNA) sequence, the DNA will zip again and (gRNA) will continue searching for a PAM sequence.
Cas9
CRISPR-Cas9
How does CRISPR-Cas9 target specific gene and bind with it?
Targeting and Binding are significant events that occur through a specific process driven by cas9 protein and guide RNA (gRNA). To target a certain gene, the gRNA is specially made to carry out specific sequence of nitrogenous nucleotides that are designed to be complementary to the specific DNA sequence. The gRNA directs the Cas9 protein to the target site by base-pairing with the matching DNA sequence in the genome. This bonding will affect Cas9 3D structure. After Cas9 protein binds to the DNA, but it first needs to find a nearby sequence called a PAM. In most cases, this PAM is a short “NGG” sequence, where “N” can be any nucleotide. The presence of the PAM is essential because Cas9 will only bind to DNA if a PAM is close to the target site. Once Cas9 locates the PAM, the gRNA pairs with the matching DNA sequence. This pairing forms a stable RNA-DNA hybrid, ensuring that Cas9 is positioned at the exact spot for editing. Once properly bound, Cas9 is ready to cut the DNA at the target site.
Guiding RNA & DNA Segment
How does CRISPR-Cas9 cleave the DNA?
Cas9 enzyme’s unwinds the DNA and it’s nuclease activity is ready for taking on the process and permits into to cut the DNA. The cleaving happens at site Cas9 nuclease domain, at this region two different sites in the nuclease domain make the cuts one on each DNA strand. As the enzyme cuts down both strands of DNA at a site upstream of PAM nucleotides (three nucleotides upstream; towards the 5’ end of the PAM strand). And the cutting stopes because the target sequence is now isolated from the rest of the gene. Resulting in double strands of DNA break, and if they were not to rejoin together a mutations or a genome rearrangements could happen.
Cas9 Cleaving both DNA Strands
How does CRISPR-Cas9 repair DNA to ‘knock out’ a gene?
CRISPR-Cas9 could “knocks out” a gene by disrupting its function through error that can be made throughout the DNA repair. After Cas9 creates a double-strand break at the target site, the cell attempts to repair the break using a process called non-homologous end joining (NHEJ). This repair mechanism is often imprecise and can lead to small mutations (insertions or deletions) at the cut site. These mutations can shift the reading frame of the gene, leading to a frameshift mutation, or introduce premature stop codons, which result in a stop, which are now considered non-functional protein. This disruption effectively “knocks out” the gene, disabling its normal function.
NHEJ Repair Mechanism
How CRISPR-Cas9 can repair a mutation in DNA?
Alternatively, CRISPR-Cas9 could have a positive effect. The second type of DNA repairing is Homology-Directed Repair (HDR). Homology-directed repair (HDR) is a precise DNA repair process that occurs after a double-strand break. The cell uses a identical DNA sequence, often from a sister chromatid, as a template to guide the repair. The broken DNA ends are first trimmed, then aligned with the matching template. The cell’s repair machinery uses this template to accurately copy and replace the missing/damaged genetic information, ensuring the DNA is restored/rejoined without errors.
HDR Repair Mechanism
How could CRISPR-Cas9 be used to our benefit?
In the interactive model we explored we were also able to view videos that went into details about how this tool could be beneficial in our life. CRISPR-Cas9 can be significantly used for many purpose due to it’s property of being the source of allowing genetic materials to be inserted, removed, or altered at particular locations in DNA genes which are very important changes in an organism’s genetic code. CRISPR-Cas9 can benefit us in numerous ways, particularly in healthcare, agriculture, and research. In medicine, it holds the potential to treat genetic disorders like sickle cell anemia, cystic fibrosis, and muscular dystrophy by directly correcting faulty genes. It can also be used in cancer treatment to target and modify specific mutations that drive tumor growth. In agriculture, CRISPR-Cas9 can create crops that are more resistant to diseases, pests, and environmental stress, improving food security. Additionally, in research, it allows scientists to study gene function with greater precision, accelerating our understanding of genetics and disease pathways. It facilitate the manipulate genomes of organisms in scientific experiments to allow for new discoveries and breakthroughs. This tools could be used in the processes where scientist are analyzing genes to modify certain characteristics of important crops and animals.
In what ways did the model accurately reflect/misrepresent the process and what improvements could be made?
The BioInteractive model we’ve used to learn about CRISPR-Cas9 accurately demonstrated the different steps in each stage of the process, in a very engaging way and more simplified descriptions at each step where an event is occurring. This simulation model reflected the big idea in a few steps which made it easy for us to understand what is happening in order. The use of labels, variety of color to draw our attention to each point was a very good method, the use of color made it simple for us to see the different parts of the genome and made is easy to visualize the process taking place. On the other hand, we know from research and reading about CRISPR that sometimes the guide RNA could target and incorrect sequences where the DNA rezip and (gRNA) would continue to look for the complimentary sequence for the wanted gene, but this model made it that gRNA travels straight to the desired gene which is a misrepresentation of the process for students. To further improve the model to be a more efficient tool to learn from would be to add more complex steps. for example there would be two paths a student would have to explore both: one would be where gRNA would target a non-complimentary sequence and the model would show the rezipping of DNA helix. Alternatively, the second path would show the gRNA looking for it’s complimentary sequence again. Also, at the cleaving of the DNA there is many events taking place like the “Kick-in” and “Kick-out”, the model revealed all process taking place in way that it all happened at once. To improve on that the model could’ve been more clear of each event in order, a zoom into the process would’ve made it simpler to comprehend. Similarly, the paper-cut is very efficient method for students to get an idea of what’s happening, but it lacked lots of important information one what’s going on, for me personally I didn’t realize what was happening until I explored the digital activity. It also didn’t not show us the 3D dimensional structure. Overall, models either digital or step-by-step paper cut out activity both are effective to educate the scientific concepts to non-scientific audiences. Even though, there could be some missed crucial elements, I still think that these models have the basic concepts in a simplified matter to teach others about the big idea. The only exception where these models could not be a good tool, is that based on the person’s learning techniques. In other words, people learn in many different ways, and for these models if they were sufficient for one person that could not be the case for the rest as people learn depending on their learning strategies. But the simulation model along with the paper model both would still be good methods for the over view of the scientific tools for majority of audience.
Work Cited:
CRISPR-Cas9 paper model in class activity handout
https://www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications