CRISPRed | A FOK1 TALE of the unexpected tools for genome editing

CRISPRed | A FOK1 TALE of the unexpected tools for genome editing

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
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

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