3 answers
Tanish
Student
Sunnyvale, California
1
Question
Asked
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How does CRISPR work?
I am a tenth grade student interested in learning more about gene expression and modification as well as protein synthesis.
#genetics #CRISPR #biology #molecular biology
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3 answers
Zachary Su
Corps Member
1
Answer
San Francisco, California
Updated
Zachary’s Answer
Hi Tanish,
First of all, props to you for being proactive about learning about CRISPR-Cas9. It is a revolutionary system of gene-editing that has changed the field of molecular biology. CRISPR was discovered first as a way by which bacteria develop immunity to infections by viruses. It was then repurposed by researchers Jennifer Doudna and Emmanuelle Charpentier for gene-editing.
The CRISPR-Cas9 system can be distilled down to two parts: 1) a single-stranded guide RNA and 2) the Cas9 protein. You can think of the RNA as half of a strand of RNA with base pairs exposed, and you can think of the Cas9 protein as a pair of molecular scissors. The two parts must form a complex and be introduced into the cell in some way. Inside the cell, CRISPR-Cas9 scans the DNA (genetic material). It opens the DNA and tries to find a sequence of base pairs that is complementary to the sequence of the single-stranded guide RNA. Once the sequence is found, the RNA anneals to the complementary sequence, bringing Cas9 to the site. Cas9 has two catalytic sites (parts of the protein at which chemistry happens). Each site will cut one strand of the DNA (remember DNA is double-stranded). As a result, a double-stranded break is created (pretty much the DNA is broken in half). The power of CRISPR is that you can control the sequence of the guide RNA. You can synthesize any RNA sequence pretty easily and quickly in the lab, which gives the CRISPR system much more efficiency compared to systems like zinc-finger nucleases and TALENS which require you to synthesize chains of proteins (which is much harder).
Ok, so at this point we pretty much can create a double-stranded break in the DNA at a location of our choice. Now, how does this allow us to make gene edits? As Mansi was saying, we harness the cell's DNA repair machinery to ultimately generate edits in the DNA. So if we want to delete a gene, we will rely on the non-homologous end-joining (NHEJ) mechanism. NHEJ is pretty much consists of jamming the two broken ends of the DNA back together. It is very error prone and usually incorporates small insertions or deletions (indels). Indels can result in the termination of the production of a protein because these small mutations shift the codon reading frame. This process is a bit complicated to explain, because it involves stepping through the transitions from DNA to RNA to protein. If you haven't learned it already, I would ask a biology teacher. Ok, so now we know NHEJ creates indels which will result in protein deletion.
Now, what if we want to fix a broken gene (e.g. change the sequence of an existing gene)? In this case, we will rely on the homologous-directed repair (HDR) mechanism. So in this process, the cell will recognize the double-stranded break created by Cas9 and search for another strand of genetic material similar to the sequence close to the break. It will then use that genetic material as a template to guide the repair process for the broken strand. Cells sometimes have multiple copies of the same DNA. When this is the case, the broken DNA will be repaired such that the original sequence is restored. However, if you introduce a donor DNA template (which consists of the final sequence of the gene you want - with the edits, along with flanking homologous arms - these match the sequence of the DNA surrounding the double-stranded break) then you can encourage the cell to incorporate the sequence of the gene you want into its DNA. There are ways of increasing the likelihood of this relatively rare process (one of which is tethering the DNA donor template to the Cas9 complex). So you have learned that CRISPR-Cas9 can create gene deletions and alterations by catalyzing a double-stranded break and relying on DNA repair mechanisms.
Now for your broader question about how to learn more about the central dogma (gene expression and protein synthesis). First of all, make sure you have the central dogma down. Know that DNA --> RNA --> protein and the various manipulations between these forms. If you are unsure about this process, there are various YouTube videos you can watch (see one below). Next, I would read about and watch some videos on gene expression systems. Some examples are lac operon and Cre-lox. You can Google these for descriptions. For CRISPR-related resources, I would check out Jennifer Doudna's TED talk and if you would like a better description and insight onto to how people design CRISPR experiments, look at Addgene. Finally, while reading and watching videos is useful, I believe that hands-on experience will be the most beneficial for your learning. If a university or research lab is close by, maybe try emailing professors there and see if you can land an internship or volunteer position working in a molecular biology lab. That is the way I learned what I know about CRISPR. Hope this helps, and good luck on your journey.
Watch molecular animation of Central Dogma: https://www.youtube.com/watch?v=9kOGOY7vthk.
Watch Jennifer Doudna's TED talk: https://www.youtube.com/watch?v=TdBAHexVYzc.
Read Addgene's CRISPR guide: https://www.addgene.org/guides/crispr/.
First of all, props to you for being proactive about learning about CRISPR-Cas9. It is a revolutionary system of gene-editing that has changed the field of molecular biology. CRISPR was discovered first as a way by which bacteria develop immunity to infections by viruses. It was then repurposed by researchers Jennifer Doudna and Emmanuelle Charpentier for gene-editing.
The CRISPR-Cas9 system can be distilled down to two parts: 1) a single-stranded guide RNA and 2) the Cas9 protein. You can think of the RNA as half of a strand of RNA with base pairs exposed, and you can think of the Cas9 protein as a pair of molecular scissors. The two parts must form a complex and be introduced into the cell in some way. Inside the cell, CRISPR-Cas9 scans the DNA (genetic material). It opens the DNA and tries to find a sequence of base pairs that is complementary to the sequence of the single-stranded guide RNA. Once the sequence is found, the RNA anneals to the complementary sequence, bringing Cas9 to the site. Cas9 has two catalytic sites (parts of the protein at which chemistry happens). Each site will cut one strand of the DNA (remember DNA is double-stranded). As a result, a double-stranded break is created (pretty much the DNA is broken in half). The power of CRISPR is that you can control the sequence of the guide RNA. You can synthesize any RNA sequence pretty easily and quickly in the lab, which gives the CRISPR system much more efficiency compared to systems like zinc-finger nucleases and TALENS which require you to synthesize chains of proteins (which is much harder).
Ok, so at this point we pretty much can create a double-stranded break in the DNA at a location of our choice. Now, how does this allow us to make gene edits? As Mansi was saying, we harness the cell's DNA repair machinery to ultimately generate edits in the DNA. So if we want to delete a gene, we will rely on the non-homologous end-joining (NHEJ) mechanism. NHEJ is pretty much consists of jamming the two broken ends of the DNA back together. It is very error prone and usually incorporates small insertions or deletions (indels). Indels can result in the termination of the production of a protein because these small mutations shift the codon reading frame. This process is a bit complicated to explain, because it involves stepping through the transitions from DNA to RNA to protein. If you haven't learned it already, I would ask a biology teacher. Ok, so now we know NHEJ creates indels which will result in protein deletion.
Now, what if we want to fix a broken gene (e.g. change the sequence of an existing gene)? In this case, we will rely on the homologous-directed repair (HDR) mechanism. So in this process, the cell will recognize the double-stranded break created by Cas9 and search for another strand of genetic material similar to the sequence close to the break. It will then use that genetic material as a template to guide the repair process for the broken strand. Cells sometimes have multiple copies of the same DNA. When this is the case, the broken DNA will be repaired such that the original sequence is restored. However, if you introduce a donor DNA template (which consists of the final sequence of the gene you want - with the edits, along with flanking homologous arms - these match the sequence of the DNA surrounding the double-stranded break) then you can encourage the cell to incorporate the sequence of the gene you want into its DNA. There are ways of increasing the likelihood of this relatively rare process (one of which is tethering the DNA donor template to the Cas9 complex). So you have learned that CRISPR-Cas9 can create gene deletions and alterations by catalyzing a double-stranded break and relying on DNA repair mechanisms.
Now for your broader question about how to learn more about the central dogma (gene expression and protein synthesis). First of all, make sure you have the central dogma down. Know that DNA --> RNA --> protein and the various manipulations between these forms. If you are unsure about this process, there are various YouTube videos you can watch (see one below). Next, I would read about and watch some videos on gene expression systems. Some examples are lac operon and Cre-lox. You can Google these for descriptions. For CRISPR-related resources, I would check out Jennifer Doudna's TED talk and if you would like a better description and insight onto to how people design CRISPR experiments, look at Addgene. Finally, while reading and watching videos is useful, I believe that hands-on experience will be the most beneficial for your learning. If a university or research lab is close by, maybe try emailing professors there and see if you can land an internship or volunteer position working in a molecular biology lab. That is the way I learned what I know about CRISPR. Hope this helps, and good luck on your journey.
Zachary recommends the following next steps:
Mansi Karkhanis, PhD
Field Scientific Director
8
Answers
Houston, Texas
Updated
Mansi’s Answer
(CRISPR)-Cas9 genome-editing technology has been derived from the bacterial immune system, it involves the use of a chimeric single-guide RNA (sgRNA) that can direct the nuclease Cas9 to catalyze a DNA double-stranded break (DSB) at a specific site within the genome of effectively any organism. The DSBs catalyzed by Cas9 trigger the host DNA repair pathways. The repair of the DSB by non-homologous end joining (NHEJ) can introduce mutagenic insertions or deletions (indels), whereas homology directed repair (HDR) using an exogenous DNA template can introduce new sequences at the target locus. The CRISPR-Cas9 system consists of three components: (1) Cas9, which can be supplied as a protein, mRNA or DNA; (2) sgRNA, which can be in the form of an RNA or a DNA template; and (3) a donor DNA to incorporate a marker/tag/mutation at the target locus.
https://www.youtube.com/watch?v=4YKFw2KZA5o
Mansi recommends the following next steps:
Olivia Jones
Pharmacy Intern
11
Answers
Fort Wayne, Indiana
Updated
Olivia’s Answer
Hello,
I do not have a direct answer to your question but if you are really interested in genetics I do know that my pharmacy school in Fort Wayne, Indiana has a pharmacogenomics masters degree program. It is all about how drugs and genes work together! It is a growing field and something that you can get a degree in separately or in combination with a pharmacy degree. My school is called Manchester University in case you are curious to look into this degree more!
Let me know if you want anymore information,
Olivia
I do not have a direct answer to your question but if you are really interested in genetics I do know that my pharmacy school in Fort Wayne, Indiana has a pharmacogenomics masters degree program. It is all about how drugs and genes work together! It is a growing field and something that you can get a degree in separately or in combination with a pharmacy degree. My school is called Manchester University in case you are curious to look into this degree more!
Let me know if you want anymore information,
Olivia
Thank you for the information. I found it really insightful! This seems like such an interesting major and I'll be sure to check it out. Are you majoring in this, or do you have another major? Thanks!
Tanish
Yes PGx is a new and upcoming field of study! I am a current pharmacy student in my third year of study. I am not in the dual degree program so I will not be graduating with a pharmacogenomics degree. They teach us a lot about it in pharmacy school though and it is very interesting. Manchester University is one of the few schools that has PGx right now! I believe there are a few others too!
Here is a link to my school website concerning the PGx program specifically! I believe it is even an online masters program now!
https://www.manchester.edu/academics/colleges/college-of-pharmacy-natural-health-sciences/academic-programs/pharmacogenomics
Olivia Jones