Introduction:
CRISPR-Cas9 is a biological technology in our cells that edits genes to improve our DNA. In other words, CRISPR-Cas9 changes the genomes of living organisms. In this lab, we learned how CRISPR-Cas9 works, why it modifies genes in our DNA, when it does its job, and where this is done. An example of CRISPR-Cas9 helping the human body is when it adds in a sequence of nucleotides (GAGT) to prevent the risk of heart disease. We explored the functions of CRISPR-Cas9 by learning kinesthetically with paper diagrams and learning visually with the help of an online simulation.
Here’s a picture of us with practically no knowledge about CRISPR-Cas9 (Sal is taking the picture).
What is CRISPR-Cas9 and How Does it Work:
CRISPR is an acronym for “Clustered Regularly Interspace Short Palindromic Repeats”, meaning a short sequence of DNA that is clustered, repeats itself in the sequence, and it can be read the same forwards and backwards. Cas9 is a tightly coiled protein that cuts the DNA nucleotides (like the scissors enzyme called helicase in DNA replication) at specific locations to make modifications. But, in order to know where to go, the guide RNA works as a GPS system to lead the Cas9 to the correct DNA sequence so it can edit and modify the strand. CRISPR-Cas9 targets specific genes by reading the DNA sequence from the 5′ to 3′ on the DNA backbone. Once a bad or negative nucleotide sequence is found, it notices and binds to a sequence of nucleotides called PAM, then the Cas9 will edit the sequence to improve it, and continue to read the DNA strand, repeating the process.
Here is a picture of our CRISPR-Cas9 and the guide RNA working together.
Targeting:
CRISPR-Cas9 starts the process by targeting a specific gene. The guide RNA will contain a sequence of about 20 nucleotides that matches to the particular target gene on the DNA strand. When both the guide RNA and the Cas9 come together to do their job, they will be guided to the target sequence, which is located near the PAM motif (the PAM motif is found everywhere throughout the cell’s complete set of DNA). Next, the Cas9 will unwind and pull apart the double helix of DNA in front of the PAM motif, closer to the 5′ end of the DNA strand.
Here are pictures of the CRISPR-Cas9 reading the strand of DNA from 5′ to 3′.
Binding:
The Cas9 binds and reads the strand of DNA in the 5′ to 3′ direction of the sugar and phosphate backbone. If the sequence of the unpaired DNA strand is not and exact match to the 20 nucleotide sequence with the guide RNA, then the Cas9 will disengage from the DNA and zip up the double helix to its original form. On the other hand, if the 20 nucleotide sequence is an exact match with the guide RNA, then it will form complimentary base pairs with the DNA sequence, leaving a helix of DNA and RNA.
Here is a picture of the CRISPR-Cas9 model reading the DNA.
Cleaving:
When the guide RNA and target DNA strand bind together due to the matching base pairs, the Cas9 gets activated to cut the DNA. Then, it starts to cleave both strands of the DNA and RNA helix, meaning the Cas9 makes specific cuts to the DNA strand (once again, in front of the PAM site). Two active sites of the Cas9 bind to each of the DNA strands, and they are what generate the cuts and cleaves to the open double helix of DNA, resulting in a double-stranded DNA break.
Here is an example of the Cas9 cleaving – or cutting – the strands of DNA, resulting in a DNA break.
DNA Repair:
The DNA breaks can be repaired two different ways: with a nonhomologous end joining (NHEJ), or a homology-directed repair (HDR).
NHEJ: This version of DNA repair is more common, most used, and it is a faster repair mechanism. This is because the cell is not using a template to join the broken ends of DNA back together. However, NHEJ is a process that contains errors, which can cause mutations in the target DNA sequence. When the break is repaired correctly, the Cas9 will once again recognize the error that was created in the target sequence and cleave it. The problem is that this repeated cycle of cleavage and repair can eventually result in a mutation in the gene by adding or deleting base pairs. The type of mutation can be random – meaning it can be positive, negative or neutral – but it will be happening within the target sequence and it could inactivate the gene if it is in the gene’s coding region for a protein.
identical copies of a chromosome that are joined at the centromere
HDR: The HDR process is prone to making less errors and uses a homologous DNA template to accurately repair the DNA break. An example of a DNA repair template can be an identical copy of a chromosome called the sister chromatin. Scientists experiment with this repair system by introducing the cell to more DNA repair templates, Cas9s and guide RNAs than what are needed in the cell. This method tricks the cell’s repair machinery into using the repair template to fix the DNA break with the HDR process, rather than using the NHEJ process which can cause errors or mutations. In addition, scientists can change the target DNA sequence and the DNA repair templates can correct existing mutations by replacing them with a new, non-mutated sequence of DNA.
In the end, the difference between both DNA repair systems is that one repairs the DNA break naturally and faster, but it can cause mutations (NHEJ), or the DNA repair can be done slower and not naturally, but it will be correcting and preventing mutations (HDR).
This is a picture of our repaired DNA break.
Analysis:
With the help of learning kinesthetically with paper diagrams and visually with an online simulation, a good and solid understanding of CRISPR-Cas9 was developed. For example, when working with the paper model, my group and I had got introduction into seeing how Cas9 works to target, bind, cleave, and repair the DNA, and we noticed how repetitive the process is for the protein – or how tiring it can be to slide pieces of paper through a system to be “read”. However, the paper model didn’t clearly differentiate between each step of the process, plus, we couldn’t exactly replicate the steps done by the CRISPR-Cas9 in a paper model. Then, the simulation visually represented all the steps to CRISPR-Cas9 clearly with bold colours and text that elaborated on any details that were needed. The simulation would sometimes overlap the steps, leading to some confusion in comprehension, and once again, we couldn’t physically recreate the steps ourselves. To improve each model, I would make sure the online simulation had clear step by step directives to avoid confusion, then add some colour to the paper simulation to show the different molecules involved with CRISPR-Cas9 process.
I believe models referring to the importance of CRISPR-Cas9 are extremely valuable to communicate, demonstrate, and educate to the public. If we are wanting to change the knowledge of CRISPR-Cas9 in our society from nothing into something valuable, then the best way for that to be done it by teaching our society about this process. We won’t grow our knowledge of science or DNA if we don’t start our learning about the process.
References:
CRISPR-Cas9 Mechanism and Applications Simulation – CRISPR-Cas9,CRISPR-Cas9 Mechanism & Applications
Ms. Yorke’s OneNote – https://sd43bcca.sharepoint.com/sites/AP12Spring2025P1523/_layouts/OneNote.aspx?id=%2Fsites%2FAP12Spring2025P1523%2FSiteAssets%2FA%26P12%20Spring%202025%20P1%20Notebook&wd=target%28_Content%20Library%2FCell%20Processes%2FDNA%20ProteinSynthesis.one%7C1ED46EAE-8A65-4F2B-B754-5FCEDA1FD3F6%2FCRISPR%20Edublog%7C65A857F9-F153-4853-9845-6E00B454DC40%2F%29