CRISPR/CAS9

FROM BACTERIAL ADAPTIVE IMMUNE SYSTEM TO GENE

EDITING (1)

It was discovered that bacteria had repetitive DNA sequences in their genomes called
CRISPRs (Clusters of regularly interspaced short palindromic repeats).
In bacterial chromosomes there were found repetitive sequences of DNA (see black
diamonds in figure 1) that flank unique sequences (see different color squares) that come
from viral DNA. CRISPR segments are repetitive sequences in bacterial DNA separated by
short segments called spacer DNA (similar to virus DNA sequence). Near CRISPRs, Cas
genes were close.

Figure 1. Adapted from (2)

How does it works in bacteria to protect

them from viruses?
When a virus infects a bacteria (and if the bacteria
survives to the infection), the bacteria can acquire
segments of viral DNA in its own genome forming CRISPR
arrays. Fragments of invading foreign DNA integrate within
the host CRISPR locus, these integrated fragments are
called spacer DNA and are responsible for the "adaptive
immunity". So, when another infection begins, the bacteria
use the arrays to transcribe them into a single strand of
RNA molecule. This strand of RNA broke into smaller units
that are composed of a sequence derived from viral DNA
together with another sequence derived from bacterial
DNA adjacent to the viral DNA in the CRISPR array.

These fragments of RNA bind tracrRNA (Tracr binds to the

bacterial DNA repeat derived RNA sequence). These two
molecules of RNA form a structure to bind the enzyme
Cas9. These ARN-protein complex surveys the cell looking
for viral DNA sequences that match the sequence of the
complex ARN (derived from the DNA viral sequence in
CRISPRs). When a sequence matches, Cas 9 unwinds the
infectious viral DNA to allow RNA/DNA hybridization and a
double strand broke is induced in the infectious viral DNA
and its degradation prevents virions formations.
The events are represented in figure 2.

Note: The CRISPR RNA (crRNA) is composed of two parts:

1. The spacer derived RNA. So RNA guide sequences are
derived from the spacer DNA sequences.
2. And RNA derived from the repeat sequences of
bacterial DNA in CRISPR loci in bacterial genome.

Cas proteins along with other RNA components (like

TracrRNA) recognise crRNA due to the presence of the
RNA sequence derived from the bacterial DNA repeated
sequences in CRISPR loci.

The crRNA recognizes complementary sequences in

invading DNA and base pair with these molecules, which
initiates cleavage of the foreign DNA molecule.

Types of systems
CRISPR systems are classified according to the numbers
and types of Cas proteins found in the systems.

Class I system include multiple genes and multiple Cas

proteins. And are more difficult to harness these protein
for gene editing.

Class II system include a single genes encoding one large

protein that combines with CRISPR ARNs. Are easy to
harness. This system is the one that uses TracrRNA that
binds CRISPR ARN to form a single guide form to Cas9 to
cut. The natural CRISPR ARN (the one derived from from
bacterial DNA adjacent to the viral DNA in the CRISPR Figure 2. Adapted from (2)
array) and TracrRNA are combined linked together
covalently making a single transcript.

The systems only differ in the mechanism that produces

CRISPR RNA and Cas proteins.
 Cas9 protein
Is an enzyme that recognise double stranded
DNA at positions in the DNA sequence that
match 20 nucleotide sequence in the single
stranded ARN guide. And makes double
strand DNA cuts.
It unwinds DNA without ATP or GTP use. This
is done by conformational changes when
binding to nucleic acids.
HNH domain is one of the chemical clivers of
DNA. And it is thought that the non target
strand of DNA must be present in the
complex in order to achieve cutting.
The protein itself is a sensor of the degree of
the complementary match in guideRNA-
targedDNA sequences. When nucleotide
complementarity is full the enzyme completely
activates and cuts the double strand DNA
target.
Mutations in Cas 9 can make the protein a
better sensor in order to be more accurate in
cutting and gene editing.
In order to cut precisely the desired DNA
molecule recognition must occur between the
RNA guide and DNA target, but only if the
target DNA sequence is next to a protospacer
adjacent motif (PAM) that are close to the
recognition sequence in the DNA target
molecule.
Types of systems
CRISPR systems are classified according to
the numbers and types of Cas proteins
found in the systems.
Class I system include multiple genes and
multiple Cas proteins. And are more difficult
to harness these protein for gene editing.
Class II system include a single genes
encoding one large protein that combines
with CRISPR ARNs. Are easy to harness. This
system is the one that uses TracrRNA that
binds CRISPR ARN to form a single guide
form to Cas9 to cut.
Note: TracrRNA is transcribed from
upstream of CRISPR loci from genomic loci.
And Cas9 belongs to system type II.
Anti-CRISPRS
This anti-CRISPRS can inhibit the CRISPR Cas
pathway, by stopping CRISPR proteins, prevent
binding to RNA, etc. Produced by phages, have
genes that encode this inhibitory proteins (very
small, aprox 100 aa).
C1 is one example that blocks the DNA cutting.
They can be used to limit activity of CRISPR Cas, to
reduce activities that may be occurring at off-
target sites.
Other forms of gene editing
Zinc‐finger nucleases (ZFNs) and
transcription activator‐like effector
nucleases (TALENs) are other examples of
genome editing machines. That make
double strain DNA break. But CRISPR/Cas9
is way more simple, cheaper and effective.
Figure 2. Adapted from (2)
Why and how it is used for gene
editing
It is very simple to design single strained RNA
guide molecule, all needed to know is which one is
the DNA target sequence and by Watson-Crick
base pairing rules, a complementary RNA single
string is designed.
Every time it is used the same protein (Cas9), so it
is no needed to design new proteins each time it is
going to be used.
The CRISPR(Cas9 system is only used to guide
Cas9 to acquire desirable double strand cuts in
specific sequences of DNA, further that point of
the double break everything that happens
depends on cellular DNA repair activities.
Gene editing can help to cure diseased caused
only by mutations in the DNA sequence, like sickle
cell anemia
Subsequent DNA repair
In homologous recombination repair (HDR)
mechanism, a donor DNA that is provided by the
experimenter a(that has the modifications wanted
for the homologous sequence in cell target DNA),
serve as a template for repair for the double
strand break, and sequence repair then could
include the modifications wanted by the
experimenter. (3)
It is important that the donor sequence for
homologous repair has light changes on it, in
order to prevent re cut by Cas9 complex, changes
in the recognition sequences for example
(mutations in PAMs could work).
the ends of the break start searching for
homology, either in a sister chromatid (like in
recombination in meiosis), or in an homologous
chromosome, or look for the donor DNA
homologous sequence.
so is needed to have homology in the donor to al
least one side of the break (minimum for things to
work fine).
Non homologous join repair (NHEJ) mechanism
could be used by cells, and it is a panic response
(there is not an homologous sequence to use as a
template), because the cell prefers to jam in the
ends of the broke DNA rather than lose the
complete segment. and this end direct joining
could make mistakes, like localized insertions and
deletions, and produce targeted mutations (small
insertions and occasionally larger insertions. And
deletions with microhomologies that can be
recognized because the sequence that is at the
junction could be assigned either to the left side or
the right side of the junction). Deletions that are in
coding sequence could be an in frame deletions or
out or frame deletions depending if the number of
nucleotides deleted are multiple of 3 (in frame
deletions) or are not multiple of 3 (out of frame
deletions). (3)
 Aplications
Genome wide CRISPR
With CRISPR/Cas9 technology, it can be produced knockout (by
libraries
NHEJ), knockin (by HDR) and knock off animal models. As well it
can be generated site specific mutagenesis (by NHEJ) and There are three of them:
corrections (by HDR). 1. CRISPR knockout. Is a loss of function
The system of gene editing is cheap, easy to manage, has a lot method, identifies new biological
of success and it is applicable to every form of living cell with mechanism.
DNA genome. 2. CRISPRa activation. A gene activation
approach ised in screening the gain of
In cancer can be used to enhance ex vivo lymphocytes that are function.
more efficient killing tumor cells once introduced again in the 3. CRISPRi inhibition. A gene inhibition
subject. Or can be used to generate mutation in cancer cells in that is used in screening the gost for
order to kill them. Can help to find knew immunotherapy two main loss of functions.
targets for cancer and other diseases.

It can be used to cure illnesses produced only by DNA

mutations. And can be a knew way to treat and cure
neurodegenerative diseases.

Can be used to alter the HIV genome and inactivate expression

of HIV genes, and make immune cells to the HIV infections.

It can be used as an antimicrobial agent, to kill antibiotic

resistant bacterial strains. It can cleave antibiotic resistant
genes. And in agriculture can be useful too.

The SHERLOCK technique is developed with CRISPR Cas, it

consist of identification of specific RNA or DNA sequences by
using fluorescent molecules attached to Cas9.

And helps in the development of synthetic biological circuits.

REFERENCES
1. Khanzadi MN, Khan AA. CRISPR/Cas9: Nature’s gift to prokaryotes and an
auspicious tool in genome editing. J Basic Microbiol. 2020;60(2):91–102.
2. Jennifer Doudna: CRISPR Basics - YouTube [Internet]. [cited 2021 Apr 4].
Available from: https://www.youtube.com/watch?v=47pkFey3CZ0
3. Dana Carroll: Background on Genome Editing - YouTube [Internet]. [cited 2021
Apr 3]. Available from: https://www.youtube.com/watch?v=upzSmpnxNig

MADE BY : JUAN FELIPE DUARTE ZAMBRANO

MAIL: JDUARTEZ @ UNAL EDU CO. .

04/04/2021