We've learned about a few techniques in biotechnology already, but the CRISPR-Cas9 system is one of the most exciting ones. Inspired by bacterial immune response to viruses, this site-specific gene editing technique won the Nobel prize in chemistry in 2020, going to Jennifer Doudna and Emmanuelle Charpentier. How did they develop this method? What can it be used for? Let's get the full story!
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We’ve examined a handful of biotechnology concepts
in previous tutorials, but now it’s time to introduce what is undoubtedly the most promising
technique in biotechnology of the past decade. The CRISPR-Cas9 system represents genome editing
technology that has revolutionized molecular biology, due to its precise and site-specific gene
editing capabilities, which essentially allow for an unprecedented level of control in manipulating
the genetic information of a living organism. How does this
work mechanistically, and what are
its applications? Let’s get a closer look now, starting with some historical context.
In 1987, Atsuo Nakata and his team of researchers from the Osaka University in
Japan first reported the presence of Clustered Regularly Interspaced Short Palindromic Repeats,
abbreviated as CRISPR, in the Escherichia coli genome. These refer to short, repeated sequences
of DNA nucleotides found within the genome of prokaryotes. These sequences are the same
when read from
5' to 3' on one strand of DNA and from 5' to 3' on the complementary strand, and
are therefore described as palindromic repeats, just the way that we refer to words like racecar
or kayak as being palindromes, because they are the same whether read forwards or backwards. This
was further reported in both Gram-positive and Gram-negative bacteria, along with archaea,
leading to the obvious question regarding the relevance of CRISPR to these organisms,
which drove research for some time. Later
on, in the mid 2000s, the functionality and importance
of CRISPR was first realized in prokaryotes. As it turns out, the CRISPR system is a key component
of their adaptive immunity, which protects these prokaryotes from attack by viral DNA,
bacteriophages, and plasmids. That’s right, it may seem incredible, but even unicellular bacteria
have a very basic immune system. Recall from our studies in the immunology series that adaptive
immunity refers to the immunity that an organism acquires a
fter exposure to an antigen, either
from a pathogen or vaccination. Vaccination, for example, results in a form of adaptive
immunity in humans, since the body is exposed to antigens, and forms antibodies in response, which
contribute to the development of the immunity. The way this works for bacteria is as follows. The
unique sequences that are nestled in between the palindromic repeats, which are called spacers, are
bits of DNA that are foreign, and do not belong to the bacterium, but inst
ead originate from mobile
genetic elements, or MGEs, such as bacteriophages, transposons, or plasmids that have
previously infected the prokaryote. This was revealed by sequencing the spacers found
in the CRISPR system, which led to the hypothesis that this could be a defense mechanism employed
by bacteria to recognize foreign DNA elements. During a viral infection, bacteria acquire
a small piece of the foreign viral DNA, and integrate it into the CRISPR locus to
generate CRISPR arrays. Th
ese consist of duplicate sequences, which are the palindromic
repeats belonging to the bacterial genome, flanked by variable sequences, or spacers, which
again are from the foreign genetic elements. In this way, bacteria retain a memory,
so to speak, of a past infection. So although it was initially revealed as a
genomic component of bacteria and archaea, CRISPR has inspired a method of genome editing
that can be applied to various eukaryotic species. But before we get there, we first have
to
understand the function of CRISPR in prokaryotes, because understanding the mechanism of its natural
function will be necessary in order to understand the way it is exploited to achieve genome editing
capabilities in humans and other organisms. Let’s take a look at a particular Streptococcus
bacterium which is being attacked by a bacteriophage. Once the viral DNA is injected into
the cell, a section of it can be incorporated into the bacterial genome, and as we mentioned, it
will be ins
erted between the repeated palindromic sequences. This will now be called a spacer.
So here we can see three different spacers, potentially from three different viruses,
sandwiched in between the repeated palindromic sequences. Now we have what is called a
CRISPR array. This CRISPR array can undergo transcription, to form CRISPR RNA, abbreviated
as crRNA, although this longer strand is called pre-crRNA. Then the protein Cas9 gets involved.
Cas refers to CRISPR-associated nuclease protein, a
nd as we know, nucleases are enzymes
that are capable of cleaving DNA at specific nucleotide linkages, kind of
like a pair of scissors. In particular, Cas9 is one of the nucleases found in Streptococcus
pyogenes, which is one of the most extensively researched and characterized CRISPR-associated
nuclease proteins, so this is the one we will be looking at here inside this bacterium.
Now along with Cas9, there are also molecules of tracrRNA. These have sections
that are complementary to and t
herefore can anneal to the palindromic repeats. So for
each spacer and palindromic repeat, we end up with a complex consisting of that segment of
pre-crRNA, a tracrRNA, and a Cas9 protein. Then another enzyme called ribonuclease three,
or RNase III, will cleave the strand in between these complexes, leaving us with individual crRNA
complexes which we can call effector complexes. With these effector complexes formed, the cell
is now ready to defend against the invader whose genome produced t
hat crRNA. If this
complex encounters a section of viral DNA that has a sequence which is complementary to this
crRNA, the nuclease enzyme will coordinate, and if it recognizes a short sequence unique to the
viral genome called a protospacer adjacent motif, or PAM, then it will snip both strands of the
DNA, just a few base pairs upstream from the PAM. In doing so, it will neutralize the
virus, because its genome can no longer be transcribed properly to create more
viral particles, so infec
tion is impossible. So that gives us a reasonable understanding of
how CRISPR is employed by prokaryotic organisms as a natural defense. Now it’s time to understand
how this phenomenon came to serve as the basis for biotechnological application. This begins in
2012, when Jennifer Doudna, a molecular biologist from the University of California, Berkeley
along with French microbiologist Emmanuelle Charpentier, were the first to propose
that the bacterial CRISPR-Cas9 system could be used as a
programmable toolkit for
genome editing in humans and other animal species, and they eventually received the Nobel
prize in chemistry for their work, in 2020. So how can genome editing be achieved using this
method? The first thing we need to understand is that in bacteria, the crRNA and tracrRNA are
separate molecular entities. The first major breakthrough arrived when it was realized that
the roles of these molecules could be combined into a single molecule by fusing them together
with a
linker to generate something called single guide RNA, or sgRNA, which
can be synthesized in the lab. If the sgRNA complexes with a Cas9 protein, this
two-component system will be able to cleave DNA just as the three-component system does in
bacteria. What this means was that it was then possible to determine any sequence of about 20
base pairs as a target for editing, and all that has to be done is to synthesize the appropriate
sgRNA with the complementary sequence, and insert that into a
cell along with the Cas9 protein which
has been sourced from Streptococcus pyogenes. The complex will form, read the DNA until it
finds the appropriate sequence along with a PAM sequence, binding will occur, and DNA will
be cleaved at precisely the desired location. Cas9 has two domains, and each one
will snip one of the DNA strands. After the incision is made, the natural DNA
repair mechanism is enacted for the target DNA. The cleaved dsDNA can undergo repair via two
routes. Either by hom
ology-directed repair, abbreviated as HDR, or by non-homologous end
joining, abbreviated as NHEJ. The NHEJ pathway repairs double-strand breaks in DNA by directly
ligating without the need for a homologous template, which means a DNA strand with similar
sequence that can act as a template. The NHEJ mechanism can also introduce insertion or deletion
of specific sequences at the joining ends, thus creating what are referred to as indels.
Indels are DNA strands with either an insertion or dele
tion of nucleotide sequences. Thus, NHEJ
produces DNA strands with non-uniformity in size. The other route of repair, the HDR pathway, is
commonly found in bacterial and archaeal cells, while the NHEJ pathway we just discussed is more
common in a eukaryotic domain. The HDR process, although more complex than NHEJ, uses a homologous
DNA template. The homologous DNA template has homology to the adjacent sequences surrounding the
site of cleavage to incorporate new DNA fragments. The template
guides the repair process, and
lowers the possibility of errors. Since there is no insertion or deletion of nucleotide sequences,
the HDR pathway maintains uniformity in the size of the resulting dsDNA, unlike NHEJ.
So that covers the mechanism of CRISPR genome editing technology. Now we move on
to the potential applications, which have only expanded ever since Doudna and Charpentier
suggested the possibility of using CRISPR for genome editing in humans and other animals. The
potential scop
e of application of CRISPR is vast, and includes its use as a genetic screen
to identify genes in different cells. One of the most prominent applications is in
cancer immunotherapy. In this practice, immune T cells, which are a type of white blood cell that
fights against a disease, are genetically modified using CRISPR technology. Specifically, these
T cells are extracted from the patient’s body and modified to make them more specialized in
recognizing cancer cells and killing them once th
ey are reintroduced into the patient’s body.
Similarly, CRISPR has also found its application in therapeutic management of acquired
immunodeficiency syndrome, or AIDS, which is caused by human immunodeficiency virus, also known
as HIV, as we covered in the microbiology series. Conventional anti-retroviral therapies are
capable of suppressing viral replication. But once the virus gets converted to its proviral
form, conventional therapies are ineffective in targeting the virus. The provirus r
esides
within the immune cells and continues to make copies of itself using the immune cell machinery,
and the immune cells fail to target the proviral latent reservoir which presents the risk
of viral rebound or relapse of the disease. Other than cancer and AIDS, CRISPR has also found
immense application in developing assays to detect SARS-CoV-2 infection, the cause
of the current global pandemic. Although genome editing of human embryos
and their implantation into a human womb, as well a
s genetic editing of somatic cells, have
wide ethical concerns and potential risks, CRISPR has the promise to cure various diseases and
prevent the inheritance of gene-linked diseases. Additionally, genome editing in
plants using CRISPR technology introduces the possibility of making
plants resistant to certain diseases, improving their phenotype or observable
characteristics, incorporating certain specific traits, improving crop yield, and so
forth. With so many invigorating possibilities
for this exciting new technology, it will be
fascinating to see which of these major diseases and issues will be solved first, signaling
the dawn of a new era in molecular biology.
Comments
How far we've come in just my, as yet short, lifetime. As a teenager they were still mapping the human genome. Now we've figured out how and where to edit that genome to solve diseases. Caution is warranted, but I wish more people were impressed and inspired by this than afraid.
this video was so 'CRISP' and clear. absolutely loved this, thank you sooo much! Keep making such videos <3
Following you right away!. Having spent all my graduate and postgraduate studies in biotechnology, I found this very useful. Thanks, Prof👍
That video was amazing. Thanks for helping all of us as always Professor Dave.
Excellent! I was just coming up to my Clinical Genetics final and this was one of our last topics. It helped a bunch! Wish me luck. UCkszU2WH9gy1mb0dV-11UJg/Rf90XtDbG8GQ8gTz_prwAgUCkszU2WH9gy1mb0dV-11UJg/dv90XtfhAurw8gTgzar4DA
Thanks for making this video! I'm currently in high school and I'm super interested in things genetics related so I'm super grateful to have encountered this video with simple enough explanations for someone like me!
This modular explaination by you makes the biomolecular mechanism behind CRISPR look easy. Thanks!
Whoa!! That that was a lot of information! I'll have to watch this a few more times before I really get my head around it
Best explanation of gene slicing that I’ve seen so far.
I'm so fascinated.. I'm a premed and studyin to crack the first entrance exam but i never thought that everything I'm learning is so amazingly wonderful .. it's so vast .. like how do these chemicals know what to make and what to stick to..and who to interact with... it has to do with reactions.... i didn't know that things I'm learning is not just a part of a chapter but rather a part of me..part of my beloved environment... so important and fascinating .. but i don't give em the respect they deserve..
Thanks Dr. Dave your provided me an ample understanding of the CRISPR/Cas9 system and now my concept is clear
Better explanation than any so-called coaching institute. Thank you so much for making this simple 🙏
Very good explanation. Much better as the courses that I followed in my master’s degree where I needed to read tons of articles to get to this point. Thank you very much, please continue biotechs videos.
Way to go , professor dave ,never stop making videos ....
Thank you for finally making me understand the actual mechanism of the Cas9 protein. I'd love something on the pros and cons of NHEJ vs. HDR processes if you ever find the time to make a video about it!
This was a really great explanation of CRISPR-Cas9 and made it extremely easy to understand, especially with the beautiful visuals 👌👌!!
It's a technology that I couldn't understand well even if I read a related book, but after watching this video, I was able to understand Crisp's concept properly.
this is amazing, the fact that bacteria developed this over millions of years. and that humanity has understood it enough to use it to solve problems
You couldn't be clearer with this explanation. Thank you so much!
I learned of this several years ago, and am familiar with the work of Jennifer Goudna. I have a degree in Microbiology. Prof Dave has done very well covering this, and I now am learning much more.