CRISPR cas9 system II Genome Editing tool

In this video we will understand what is CRISPR/cas system. And how it is used for genome editing
Clustred 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 recognizes 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 inserts 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 comes in contact with   many viruses, so it creates a hitlist in its genome
The hitlist containing short 20 nt sequences from their viral dna
This hitlist is known as CRISPR
CRIPR family consists of segments of DNA containing short repetitive palindromic sequences  . After each repetition the bacteria inserts a small part of the viral DNA called the   Space DNA.These spacer vDNAs are used to detect and destroy DNA from 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   have 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 nt 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 synthesise the Cas9 guide RNA complex inside the desire cells. The plasmid vector genrally 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 synthesiszed. 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 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 dousble 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. This will be helpful in conditions where t
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 visualise 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

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