CRISPR-Cas9 / A&P12

CRISPR-Cas9 is a genetic engineering technique in molecular biology that allows for the DNA in cells and organisms to be edited in simple and efficient ways. It is a type of immune system discovered in bacteria. CRISPR can be used to inactivate genes and to edit the sequences of genes in a more specific way. This tool is widely used in current research because of its potential to treat genetic diseases, defeat viruses and produce better crops along with other things. CRISPR has many possible uses, including insertion of new genes for the organisms to produce useful medicines, creation of tailor-made organisms to study human diseases and help producing replacements for damaged or diseased tissues and organs. Overall, CRISPR has many important uses and is a tool very vital to current scientific research because of how developed its system is. Having explored it through various studies such as models and simulations have shown me just that.

CRISPR-Cas9 is a gene-editing technology where the CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and the Cas9 stands for a CRISPR-associated protein 9. It consists of two essential components: a guide RNA that matches the desired target gene and Cas9, an endonuclease that causes a double-stranded DNA break, consequently allowing modifications to the genome.

The structure of CRISPR-Cas9 includes components such as:

  • Cas9: a DNA-cutting enzyme called a nuclease.
  • Guide RNA: an RNA molecule that binds to Cas9 and allows it to find the target gene.
  • Target DNA: a DNA molecule that contains a “target gene” for CRISPR-Cas9 to cut.
  • Random nucleotides: nucleotides that can be inserted where the target gene is cut.
  • Donor DNA: DNA that can be used to edit the target gene in more specific ways.

Once programmed, the Cas9-RNA complex is now able to seek out a target DNA. CRISPR sequences are transcribes into short RNA sequences that are capable of guiding the system to matching sequences of DNA. Cas9 first finds and binds to a three-nucleotide sequence called PAM which occurs within the genome. PAM is short for protospacer adjacent motif, which occur approximately every 50 bases or less. Once they bind, it unwinds the DNA, if the guide RNA matches the 20 nucleotide DNA sequence next to the PAM, the guide RNA will bind to the complementary DNA strand. If not a match, the DNA zips back together and CAS9 will keep binding to other PAM sequences until it finds the matching target DNA. When the target DNA is found, Cas9 binds to the DNA and cuts it, shutting the targeted gene off. Cas9 binds to the Pam motif and then proceeds to unwind the DNA double helix and then the DNA and matching RNA will bind through complementary bas pairing until they form a DNA-RNA helix.

Once the guide RNA binds to the DNA, it triggers the Cas9 to change its three-dimensional structure and activates its nuclease activity of the Cas9 enzyme called cleaving. Cas9 always cleaves specific cuts in both strands of the DNA three nucleotides upstream of the PAM site. The regions where molecules bind to undergo chemical reactions are the two active sites on the nucleus domain of Cas9 which generate the cuts and cleave both strands of the DNA double helix, resulting in a double-stranded DNA break.

Finally, after Cas9 cleaves the DNA, cellular enzymes will attempt to repair the break. CRISPR-Cas9 takes advantage of these repair mechanisms to alter the target gene sequence. CRISPR can be used to inactivate a target gene, known as gene knockout, when the cell uses nonhomologous end joining (NHEJ), a repair mechanism that is sometimes error-prone and can lead to mutations that may inactivate a gene, to repair the DNA break. NHEJ is more frequently used as it is faster, and the cell doesn’t use a template to join broken DNA ends together. Mutations can be caused by repeated cycles of cleavage and repair from when errors are recognized. The type of mutation is random but will occur precisely within the desired target sequence and if its within a gene’s coding region, the mutation will likely inactivate that gene.

The second type of repair mechanisms is used to edit a target gene. In this case, the cell uses homology-directed repair (HDR) which is less error-prone the repair the DNA break. HDR fixes DNA using a template sequence from homologous chromosomes. Scientists can also provide a donor DNA as a template to manipulate the cell into using HDR since it’s less error prone. By designing different repair templates, scientists can change the target DNA sequence into a new sequence as well as allowing the temples to correct an existing mutation by replacing it with a nonmutated sequence of DNA.

CRISPR-Cas9 can be used to our benefit with how it allows scientists to rewrite the genetic code in almost any organisms while also being simpler, cheaper, and more precise than previous gene editing techniques. It also has a range of real-world applications such as, curing genetic diseases like cystic fibrosis and Huntington’s disease, creating drought-resistant crops, defeating viruses, and producing useful medicines.

Throughout the CRISPR modelling activity, the use of models to learn about the CRISPR process allowed for insightful hands-on learning. The models were able to accurately reflect the process by allowing students to participate in the step-by-step processes it takes to engage in CRISPR such as the targeting and binding by connecting paper pieces together, as well as the cleaving by making students cut apart the pieces and then finally with the different forms of repairing by allowing us to tape the pieces back together in various forms. Overall, the process clearly indicated an easy-to-understand way of how CRIPR works by giving us the opportunity to partake in the process. However, some ways in which the model misrepresented the process was by excluding some vital explanations which could’ve resulted in less confusion for students. Some changes that could be made to the modelling activities to make them better represent the process could be to have a more profound example of mutations within the genes and how both the NHEJ and the HDR affect them.

I believe that models are a very effective way to educate any and everyone about scientific concepts as it allows for the visual learning parts of brains to be stimulated. Having hands-on experience with concepts allows for the process to be taught in a more reassuring way to ensure that people are actually getting to see firsthand what they are learning about. Being able to visualize and create one’s own models allows for a clearer knowledge that can help with a long-lasting education. Especially 3-d models that allow for kinetic learners to completely understand a concept can result in much a better comprehension of ideas and therefore ensure a proper learning. Overall, models are extremely useful when it comes to scientific concepts as it can help the brain into fully gathering every piece of information and knowledge needed.

Sources:

webteam), www-core (Sanger. “What Is CRISPR-Cas9?” @yourgenome · Science Website, 8 Feb. 2022, www.yourgenome.org/facts/what-is-crispr-cas9/.

CRISPR-Cas9, media.hhmi.org/biointeractive/click/CRISPR/?_gl=1%2Aoyzf8c%2A_ga%2AMTY3OTA0NTYyOC4xNjk1NzY1MDUw%2A_ga_H0E1KHGJBH%2AMTY5NzIxMDQ1NC4xMC4wLjE2OTcyMTA0NTQuMC4wLjA. Accessed 22 Oct. 2023.

“Questions and Answers about CRISPR.” Broad Institute, 17 Dec. 2014, www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr.

EFPH11 – Bento Box

Attached below is my bento box for the novel Deep House by Thomas King. Clicking on the little yellow dots will bring you to various compostitions such as the writer’s style, other connections to literature, some characterization and more. Clicking on the dot at the bottom left corner of the book will play an audio recording of my personal take on the novel. Enjoy!

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