The crispr cas9 system was discovered as a result of observations in the mid 2000s.
During this time Researchers who were studying bacterial sequences noticed that there were
these repetitive palindromic sequences.
So the palindromic sequences would then have these unique sequences that were flanked by
these palindromic sequences.
so the unique sequences were found to be representatives of viral genomes that the bacteria would keep
if and when it survived an infection.
so a bacteria that had a crispr system would keep these snippets of viral genomes and flank
it with these repetitive sequences, if and when it survived an infection by a particular
virus.
And so it turns out that these are adaptive immune responses from bacteria.
These repeats would be processed and it would then associate with another RNA molecule before
it can then bind this DNA cutting enzyme known as CRISPR associated and so its given this
'CAS' and it has a number, the most common one is cas9 but you can have different cas
(CRISPR associated) enzymes which are able to cut DNA.
So let's give some terminology that you encounter with CRISPR Cas9 systems.
So you come across Cas9, and Cas9 as we know, these CRISPR associated enzymes can cut DNA.
They can recognise particular DNA sequences - when the guide RNA guides it.
So then there's this guide RNA concept.
So the guide RNA is this RNA that's used in bacterial adaptive responses.
It's adapted for use in genome editing.
So in the bacteria its two separate RNA components but in the lab when it's used as a tool, it's
combined into a single RNA, guide RNA (sgRNA) that's about 100 base pairs of sequence that
directs Cas9.
So cas9 binds to it, it binds to this guide RNA (gRNA) and the guide RNA in turn, binds
to one strand of double-stranded DNA.
The first 20-21 base pairs of the guide RNA (gRNA) is what gives it specificity as to
which DNA segment its going to bind.
And that's where you get to program it and tell cas9 the 20 base pairs in your genome
that you want cas9 to bind to.
And then cas9 bound to that will edit that sequence.
3 base pairs from the PAM closest to that sequence.
So i'm going to show these examples using Sickle Cell Anemia as the genetic disease
model that we may want to intervene in by designing guide RNA (gRNA) to fix the genetic
defect.
So let's have a look at how we'd go about something like that.
The first step is to go to NCBI entrez and there you can search for the gene that's responsible
for the disease.
the sickle cell disease comes about where in the haemoglobin subunit beta you have 2
thymine (nucleotides) instead
of
adenine (nucleotides).