genome editing has been going on for a
lot longer than you've probably realized
for example in agriculture humans
intervene to accelerate the rate of
editing by practices where we select for
more desired properties in plants and
animals in 1927 published in the highly
reputable journal Science
HJ muller reported in a way of appreciably
increasing the rate of changes in
genomes with this paper titled
artificial transmutation of the green
twenty years later
Charlotte aubach in 1947 also reported
in the journal Science that you can
change in ohms using radiation or
chemicals her paper was titled a
chemical production of mutations so by
1947 we realized that we can change
genomes but at this point the changes
are random and we have no way of making
targeted changes enter Mario Capecchie
and colleagues in the 1980s where they
devised gene targeting techniques
Capecchie and his team introduced
sequences with desired eclair ties into
mice that they hoped would be recombined
while homologous recombination
homologous recombination: yep, it is both
a mechanism for fixing double-stranded
breaks in genes as well as used during
meiosis to create genetic diversity by
swapping segments of homologous
chromosomes passed on by each parent
this work, which earned Capecchie a Nobel
Prize in medicine or physiology in 2007
involved using a positive and negative
selection marker on a vector that
contains the changes you wish to
introduce. It also needed to be performed
in embryonic stem cells. Using this
approach the first knockout mouse was
created in the 1980s
the technique was used in mice for
decades - very effectively - but was low in
efficiency what became apparent in those
days as Dana Caroll the genome editing
pioneer and guru at whose digital feet I
sat to learn all of these fascinating
tales: was that if there was a double
strand break in the target, the
recombination event would happen much
more efficiently. So how do we make a
double-stranded break in a targeted
manner? let me introduce you first to
zinc fingers. zinc fingers were first
discovered in 1983 with 
structures being solved in night in 1991
and  1993.  zinc
fingers are proteins known as
transcription factors that bind DNA RNA
proteins or other small molecules the
initial discovery was in the
transcription factor of frog eggs where
9 of these DNA binding proteins
contain zinc fingers are arranged it was
subsequently realized that zinc fingers
are found not only in frog frog eggs but
is found in all plant and animals they
also function in DNA recognition. Zinc
fingers later went on to become a
technology for editing DNA. But, how? Well,
Kim cha and Chadron Segura natal
published a paper in 1996 in PNAS where
they had realized that a particular
restriction enzyme known as FOK1 was
promiscuous. *scandalous*lol this means that
while most restriction enzymes are very
faithful to a particular sequence and
will only cut the DNA sequence where
those sequences are present, they found that
FOK1 was not rolling that way mm-hmm
fok1's DNA recognition and cleavage
domains were physically separable so
Chandrasegaran's group thought,
rightly, that if we can separate fok1
we can put other recognition domains on
it. the recognition domains they chose
were zinc fingers naturally occurring in
eukaryotic DNA binding transcription
factors as just mentioned. so by
designing zinc fingers that target your
DNA of interest you can use fok1
cleavage domain to cut them and fok1
would just cut any DNA that you've
told it to cut. so by designing zinc
fingers that target your DNA of interest
you can include fok1 cleavage domain
to them and fok1 will cut the DNA
the nuclease domain of fok1 has to
dimerize. 
the next technology for making
double-stranded breaks came in the form
of transcription activator like
effectors or TALEs
these are modular proteins that can also
read the sequence of bases so the
adenines, the guanines, thymines and
cytosines in DNA, they can recognize
it. They were discovered in bacteria that
infects plant specifically in
Xanthomonas bacterial species in nature
plant bacterial pathogens use these
proteins to make plant cells more infectious
by sending the proteins to the
nucleus of plants and activating
relevant genes *sneaky!* TALEs
unlike zinc fingers can bind one
nucleotide at a time, so they're easier
to work with than zinc fingers
researchers engineer these to allow them to bind any DNA sequence you want
by fusing it once again to the DNA
cutting domain of the nuclease fok1 to
allow targeted DNA editing. The next
technology to mention is the CRISPR
Cas9 system *yay*. so today we have the
CRISPR Cas9 system. this system we've
become aware of quite recently. In the
mid-2000s several researchers had come
across repetitive sequences that are
palindromes in bacterial. clarification
palindromic sequences means that it
reads the same from the front as well as
if you're reading from the back, it'll also
be the same sequences and the repetitive
sequences are flanked by unique
sequences. researchers were really
puzzled by these. The palindromic
sequences mean that those sequences
could fold and base pair with itself
resulting in structures that are quite
different from other bacterial sequences.
the palindromic sequences get
transcribed along with snippets of
unique
sequences which were not really
understood. well to cut a reasonably long
story short they were later understood
to be viral sequences that the bacteria
would keep if, and when it survived an
infection from a virus. these sequences
became known as Clustered Regularly
Interspaced Short Palindromic Repeats or
CRISPR for short and it turns out that
it's a form of natural bacterial immune
adaptive immune system where small
sequence representations of viral
genomes are kept in the bacterial system.
these representations of viral sequences
get copied to RNA, processed and
associates with another RNA molecule
called
tracrRNA before it can then bind a DNA
cutting protein known as CRISPR
associated or Cas9 for short
the most common Cas protein that is
used is Cas9 which is obtained from the
bacteria streptococcus pyogenes. Guided
by the virus sequence Cas proteins can
cleave and inactivate viral sequences. we
have adopted this technology this system
as a technology in the research
community to target specific genomic
sequences for studies. Note that the
synthetic CRISPR Cas9 tool that
is used in the lab is simplified by
linking the tracrRNA with the crispr
RNA (crRNA) and it's called a single guide RNA
or sgRNA for short
so there you have it there are three
technologies or tools you can use to
make targeted DNA double-stranded breaks
targeted double-stranded breaks in a
genome that you wish to edit. now an
important point here is that all that
you do with these technologies is make
double-stranded break and then you rely
on the cell's ability to fix
double-stranded break to cause the
changes that you want. now if the cell
uses non-homologous end joining (NHEJ)... there
are two types of ways your cell can fix
double-stranded break if you look at DNA
repair mechanisms you can you experience
all sorts of the DNA damage and there are
dedicated pathways for dealing with that:
 if the damage that you
sustain is a double-stranded break,
you have two mechanisms for fixing it in
your cells - in eukaryotic cells. so the two
mechanisms are non-homologous end
joining (NHEJ) or homology directed repair (HDR).
so once you've caused this
double-stranded break you now rely on
these two
to create changes in the genome that you
wish to see non-homologous end joining
is a panic response occasionally making
mistakes that we rely on to knock out
genes. the mistakes are frequently
localized small insertions or deletions
Homology Directed Repair you wish for the
cell to replace a particular gene
segment with the gene that you wish for
it to change. this repair mechanism happens
only at specific points in the cell cycle
and is very difficult to get so the
efficiency is much much lower. Okay so we
have a fairly easy tool, thanks to the
CRISPR Cas9 system for editing -
amazing!  but Stanley Lei Qi had an
interesting question while working in
Jennifer Doudna's lab. Jennifer Doudna is
one of the pioneers the main
pioneer of the CRISPR Cas9 technology.
while Stanley Lei Qi was working in
Jennifer's lab he asked: well what else
can we use this tool for besides cutting
DNA? catalytically dead Cas9 or dCas9
for short, allows you targeted
editing at the transcriptional level
this means that the changes you make are
not permanent because the change is not
in the DNA code itself, as in the genome
but in the messenger RNA that will be used
to make a protein. ok so let's summarize:
zinc fingers fused to fok1 nuclease
allows you to edit sequences at least
two to three base pairs at a time fok1
fused to Transcription Activator
Like Effectors TALEs allows you to edit in
increments of one base at a time the
CRISPR
Cas
system commonly Cas9, allows you to
edit any sequences as long as you tell
the CRISPR associated enzyme Cas9
or whatever enzyme that is CRISPR
associated that you have, which sequence
to cut using an RNA guide. All the best
with your experiments
