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