In this video, we will understand what is
the CRISPR/Cas system.
And how it is used for
genome editing
Clustered regularly interspaced short palindromic
repeats (CRISPR) are DNA sequences are derived
from viruses
There are two main components of The CRISPR
cas9 system: The cas9 protein and the guide
RNA
The Cas9 protein is a nuclease i.e. molecular
scissor that can cleaves nucleotide sequences.
The guide RNA, as the name suggests, guides
the Cas9 protein to make a specific cut.
The guide RNA binds to a complementary DNA
sequence, after which the cas9 nuclease makes
the cut.
We will look at a little mechanism of the
same later.
So when the virus injects its DNA into the
bacteria, the bacteria recognize the viral
DNA due to the presence of DNA markers i.e.
PAM (protospacer-adjacent motif).
Cas9 enzyme finds complementary sequences
in the viral DNA, bind to it and cleaves the
Viral DNA
The bacteria insert a part of the cleaved
viral DNA into its own genome.
So next time when the same virus comes in
the bacteria will quickly respond and cut
the viral DNA using the cas9 protein.
This is how the bacteria preserves the memory
of the invading virus.
During its lifetime the bacteria come in contact
with many viruses, so it creates a hitlist
in its genome
The hitlist containing short 20 nucleotide
sequences from their viral DNA
This hitlist is known as CRISPR
CRISPR family consists of segments of DNA
containing short repetitive palindromic sequences.
After each repetition, the bacteria insert
a small part of the viral DNA called the Space
DNA.
These spacer vDNAs are used to detect and
destroy DNA from the similar virus during
subsequent infection
Mainly cas9 protein is extracted from S.aureus
or S.pyogenes.
S.aureus Cas9 proteins are smaller than S.pyogenes
Cas9 proteins.
Both have similar On target effects, however,
S.aureus Cas9 has lesser OFF target effect.
The scientists first select a gene they want
to target and then design A short synthetic
GRNA consists of 2 parts.20 nucleotide complementay
sequences for the target DNA and a scaffold
sequence to bind to Cas9 protein.
The target site can be easily modified by
changing the complementary spacer sequence
in gRNA
A plasmid vector is created to synthesize
the Cas9 guide RNA complex inside the desire
cells.
The plasmid vector generally consists of a
promoter, cas9 sequences, a selectable marker,
ori, complementary sequences of the target
DNA, and gRNA scaffold that will bind to cas9
As the plasmid is expressed Cas9 protein and
gRNA is synthesized.
The cas9 protein has +vely charged groves
that attract the negatively charged nucleotide
sequences in the gRNA scaffold Forming ribonucleoprotein
complex
Cas9 undergoes a conformational change after
gRNA binding and becomes active.
In its active form, cas9 can now bind to DNA
sequences
The gRNA binds to the complementary target
DNA of the host cell in the 3’-5’ direction.
After binding to the target site, Cas9 undergoes
a second conformational change
Which exposes the nuclease domains RuvC and
HNH to cleave the opposite strands of the
target DNA leading to a double-stranded break
with blunt ends
The resulting DSB is then repaired by one
of two general repair pathways:
The efficient but error-prone non-homologous
end joining (NHEJ) pathway
The less efficient but high-fidelity homology-directed
repair (HDR) pathway
The repair can result in amino acid deletions,
insertions, or frameshift mutations which
ultimately leads to the loss of function of
the target gene
Thus genes can be efficiently silenced using
the Crispr cas9 system.
It is however widely used to create knockouts.
A modified version of Cas9 can be used to
activate the gene instead of silencing it.
As we know RuvC and HNH do not have any role
in the target site binding of Cas9 gRNA complex.
Thus researchers inactivated the two domains
by point mutations to create a modified nuclease
dead Cas9 which cannot cleave the target DNA
The dCas9 molecule can now be fused to any
activating domain to express a particular
gene.
Or it can be fused to Green fluorescent protein
to visualize genes in live cells using fluorescent
microscopy
Two-step control of Cas9.
Binding to gRNA activates the DNA binding
ability of cas9 and binding to
target DNA activates the 
DNA cleaving ability
