Hi guys! My name is Heather. It's so good to see
you guys again. So welcome to Module 4!
Today we're gonna talk about CRISPR Cas9. It's an amazing and revolutionary
DNA tool that's widely used in
scientific research as well as synthetic biology. If you ever
go into STEM in the future, you're
definitely going to be hear about this
CRISPR Cas9 or even use it in your future
research. So before we start the module
please do the following two things: open
up the syllabus and read the link to
articles for Module 4 titled "Questions
and Answers about CRISPR". It talks a
little bit about what CRISPR Cas9 is
which is tightly related to today's
topic. After reading the articles please
also open up the Google Form #4.
it has some questions I would like you guys to answer during this module. So if
you have done the both things then
good job and let's get started! So for
today's modules, I was first talking
about what CRISPR Cas9 is and then I'll
talk about the history of CRISPR Cas9 and how
is it originated as well as the
important components of CRISPR Cas9
which are the guide RNA I saw as
the Cas9 enzyme.  At the end I'll
talk a little bit about how CRISPR
Cas9 is delivered to the cell's
genome. So, the first question is what is
CRISPR Cas9?
so CRISPR Cas9  is an abbreviation for
Clustered Regularly Interspaced Short
Palindromic Repeats. I know it's a long
name and I'll talk more about how this
name is derived in the next slide but
like I said before, it's an amazing gene editing tool.
It really allows the scientist to edit gene
at a specific location as well as adding a
new functional DNA at the desired
location. So, as you can imagine, this
will be very helpful for since I know
biology as well as anything related with gene editing. So where is CRISPR Cas9 originated?
It's actually defense mechanism
adapted from the bacteria against virus.
So as you guys can see in this picture,
this is a virus and this is a bacterial
cell.
So when virus attacks bacteria it
inserts its viral genome into bacterial
cell. The bacteria can actually snip
parts of viral genome and incorporate
into the CRISPR region. The name
"Clustered Regularly Interspaced Short
Palindromic Repeats" is because in this CRISPR region it
consists of these palindromic repeats as
a black rhombus as well as part of virus
genome shown in this colorful square. So
why is this helpful? When the virus
attacks again the bacteria can actually
recognize that that is not part of
bacterial genome because that virus gene
is complementary to part of the virus
gene in the CRISPR sequence. Then, the
bacteria can actually make this molecular
scissor that can actually cut the
virus genome to disable the gene from the
virus and that protects bacteria from
being infected from the virus.
So when the scientists see this
mechanism they are like "Maybe we can
really incorporate this into not only
cutting the virus gene but as well as
human genome as well" and that's how
CRISPR Cas9 was discovered. So before
I go into how CRISPR Cas9 actually works,
I want to introduce the two very
important components in CRISPR Cas9. So the first one is Cas9 enzyme. So
that is actually the scissor to cut the
target DNA and that is not active when
it's just by itself. The second
component is the guide RNA. So guide RNA
contains a sequence that are
complementary to target DNA so once
the Cas9 ends up as well as a guide
RNA, it forms it's complex that's active.
So with the Cas9  enzymes that can cut the
genome as well as a guide RNA that
actually guides a Cas9 to tell
Cas9 where to cut the genome this
complex is now active and ready to do
its job. So how does CRISPR Cas9
actually work? So after a CRISPR Cas9
complex forms, it can be introduced into a
cell's genome.
Cas9 is a molecular scissor and
guide RNA , since it has a complimentary region to the
target gene,  it can bind to the target gene. The Cas9 then cuts the target gene and formed
this broken genomic DNA. This broken
genomic DNA has a deletion of the target
gene and it can either be repaired by
the cell's own repair mechanism and form a
DNA that doesn't have the target gene or
the scientists can introduce another
functional DNA into a broken genomic
and form this final product that has the
original DNA as well as an insertion of
a new functional DNA. So in the next
slide I have a video that kind of summarizes
everything that I've been talking about.
I just thought that having animation might be
helpful for understanding the content.
within our DNA that function is taken on
by a system called CRISPR Cas9. CRISPR
is short for "clustered regularly
interspaced short palindromic repeats".
CRISPR consists of two components the
Cas9 protein that can cut DNA and a
guide RNA that can recognize the
sequence of DNA to be edited. To use
CRISPR Cas9, scientists first identify
the sequence of the human genome that's
causing a health problem then they
create a specific guide RNA to recognize
that particular stretch of A's T's GS
and C's in the DNA. The guide RNA is
attached to the DNA cutting enzyme Cas9 and then this complex is introduced to
the target cells. It locates the target
letter sequence and cuts the DNA. At that
point, scientists can then edit the
existing genome by either modifying,
deleting, or inserting new sequences. It
effectively makes CRISPR Cas9 a
cut-and-paste tool for DNA editing. In
the future scientists hope to use CRISPR
Cas9 to develop critical advances in
patient care or
even cure lifelong inherited diseases.
So as you guys can see here CRISPR Cas9
is really helpful in the scientific
research. So before I go even further into
the module I would like you guys to
answer these two questions in the Google
Form. So in the first question I'm very
curious that if you are a well-known
scientist and have access to fabulous
lab and you know have an abundance of funding what
do you want to research and achieve
using CRISPR Cas9 technique. So
here I have shown some of the things that
scientists right now are doing using CRISPR
CAS9 but please feel free to use
your imagination.
The second one is why do you think CRISPR
Cas9 is helpful in synthetic biology? Now
from Module 2 you can already have a basic
understanding about what synthetic biology is
and with what I just talked about about
CRISPR Cas9 as a genome editing
tool, I'm curious to know what do you
think? How this two technique can be
related to each other? So now pause the
video and answer the question and after
answering it, play the video again and
let's continue the module. So I hope you
guys already finished completing the
question and let's keep going. So the
last concept I would like to cover is
how can CRISPR Cas9 actually be delivered to the cell? So now I
talked about how CRISPR Cas9 works but we need to have a delivery
system to have the CRISPR Cas9 into the cell's genome in the first place.
There are many delivery systems out
there in the world and today I will talk
about three method that scientists use to
deliver CRISPR Cas9 to the cell's
genome.
The first one is a lipofection. So in
the lipofection, as you can see here,
the scientists can combine the plasmid
that encodes for Cas9 and guide RNA
with a liposome. Liposome is more
just like a vesicle so it's like a sack
with lipids in there. So basically when
the scientists combine these two together
it forms a liposome nucleic acid complex
and this complex can then be easily
taken by the cell
and the plasmid with Cas9 and guide RNA is introduced to the cells genome. The
second one is Lentiviral transduction. 
In this mechanism, again, the plasmid that
encodes the Cas9 and guide RNA can be
introduced into a 293 T cells as well as
two plasmids that encode for virus. So
when this three plasma are introduced
into 293 T cells, since it has a plasmid
that encodes for virus it can then form
this Lentiviral particle that has
the Cas9 and guide RNA. This Lentivirus
can then attack a cell and it releases
the plasmid that has the guide RNA and
sorry, guide RNA and Cas9 and this plasmid
can then be introduced into a cell.
The last one is electroporation.
So in this one, the scientist first
introduced a short electric pulse into
the cell's membrane which causes forming
pores in a cell's membrane. So again this
plasmid  that has a Cas9 and guide RNA
encoding plasmid can then be introduced
to the cells through those pores. These
cells usually need to be culture later
so all these pores can be brought back
again and have a complete cell membrane. So that
kind of concludes about today's module I
hope you guys have some fun learning
about CRISPR Cas9. So I just want
to have a brief review of what I just
talked about today in the module. So the
first thing is CRISPR Cas9 is a
revolutionary gene editing technique. It really allows
scientists to specifically cut the gene
at the desired location as well as
adding a new function of DNA into the
genome. CRISPR Cas9 is actually
adapted from bacteria defense mechanism
as I showed earlier and CRISPR
Cas9 consists of two important components:
guide RNA and Cas9 enzyme. I also
talked about the mechanism of how CRISPR Cas9 
work and at the end
I talked about three CRISPR Cas9
delivery systems. So lipofection, lentivirus
delivery system as well as electroporation.
So in this module I have two
activities that I'd like you guys to do.
So first activity is that Susan has
sickle cell disease which is a genetic
disorder caused by single DNA
letter mutation. As you guys can see here
this is students DNA and here there's a
mutation that causes student to have
sickle cell disease. So I am wondering if you
could draw the genetic editing process using CRISPR Cas9 that will
help Susan correct the genome from this
mutation to a healthy DNA strand that
doesn't have that mutation. You can
refer to the slide I had before to talk
about how its work and you can
start by drawing the guide RNA as well as the
Cas9 enzyme and how these two can form a complex
 and then at the end forming
this new healthy DNA strand. The next
activity is more like a light-hearted
fun activity
allowing you guys to use CRISPR Cas9
knowledge that you got today to
explain to me what's the meaning of this
two memes. If you have any other memes
that you think you want to share
relating to CRISPR or science please
feel free to upload it in the Google
form below.
Well thank you so much for listening to
today's module! I had so much fun talking
to you about CRISPR Cas9 and I hope
you guys had some fun too.
Feel free to email us at uofr.igem@gmail.com
we are very happy to
receive any feedback questions from you
because we always love to hear from you.
Well don't forget to complete the below
form and yeah I hope you guys had some
fun and I'll see you guys next time. Bye!
