CRISPR/CAS9
Video 1
Every cell in our body contains a copy of our genome, over 20,000 genes, 3 billion letters of DNA.
DNA consists of two strands, twisted into a double helix and held together by a simple pairing rule. A
pairs with T, and G pairs with C. Our genes shape who we are as individuals and as a species. Genes
also have profound effects on health, and thanks to advances in DNA sequencing, researchers have
identified thousands of genes that affect our risk of disease. To understand how genes work,
researchers need ways to control them. Changing genes in living cells is not easy, but recently a new
method has been developed that promises to dramatically improve our ability to edit the DNA of any
species, including humans. The CRISPR method is based on a natural system used by bacteria to
protect themselves from infection by viruses. When the bacterium detects the presence of virus
DNA, it produces two types of short RNA, one of which contains a sequence that matches that of the
invading virus. These two RNAs form a complex with a protein called Cas9. Cas 9 is an nuclease, a
type of enzyme that can cut DNA. When the matching sequence, known as a guide RNA, finds its
target within the viral genome, the Cas9 cuts the target DNA, disabling the virus. Over the past few
years, researchers studying the system realize that it could be engineered to cut not just viral DNA
but any DNA sequence at a precisely chosen location by changing the guide RNA to match the target.
And this can be done not just in a test tube, but also within the nucleus of a living cell. Once inside
the nucleus, the resulting complex will lock onto a short sequence known as the PAM. The Cas9 will
unzip the DNA and match it to its target RNA. If the match is complete, the Cas9 will use two tiny
molecular scissors to cut the DNA. When this happens, the cell tries to repair the cut, but the repair
process is error prone, leading to mutations that can disable the gene, allowing researchers to
understand its function. These mutations are random, but sometimes researchers need to be more
precise, for example, by replacing a mutant gene with a healthy copy. This can be done by adding
another piece of DNA that carries the desired sequence. Once the CRISPR system has made a cut, this
DNA template then pairs up with the cut ends, recombining and replacing the original sequence with
the new version. All this can be done in cultured cells, including stem cells that can give rise to many
different cell types. It can also be done in a fertilized egg, allowing the creation of transgenic animals
with targeted mutations. And unlike previous methods, CRISPR can be used to target many genes at
once, a big advantage for studying complex human diseases that are caused not by a single mutation,
but by many genes acting toghether. These methods are being improved rapidly and will have many
applications in basic research, in drug development, in agriculture, and perhaps eventually for
treating human patients with genetic disease.
Video 2:
CRISPR is the newly discovered revolutionary tool that would allow scientists to change at will any
DNA sequence of, presumably, any living organism in a precise manner. Unlike any other previously
developed techniques of gene editing, CRISPR is remarkably simpler, faster and cheaper.
CRISPR is part of a naturally occurring defense mechanism found in many bacteria. The bacteria use
CRISPR to specifically snip the DNA of invading viruses. CRISPR stands for “Clustered Regularly
Interspaced Short Palindromic Repeats” - a region of bacterial genome that contains short DNA
repeats with unique sequences, or spacers, in between. These spacers are derived from DNA of
viruses that prey on the bacteria. The CRISPR region is essentially a DNA library of all enemies that
need to be recognized and destroyed. After being transcribed, individual pieces of spacer RNAs form
complexes with a protein named Cas, for CRISPR-ASsociated protein. Cas is an endonuclease – an
enzyme that cuts DNA. These RNA/protein complexes then drift through the cell, looking for
matching viral DNA. If a match is encountered, the RNA latches on, base-paring with it; Cas protein
then cuts the viral DNA, disabling the virus.
1
, Scientists have isolated this system, and by designing their own spacer-RNAs, they can, in theory,
target any DNA sequences in any organism. The system has indeed worked in all organisms tested so
far. The current CRISPR system consists of two components: a guide RNA and a Cas protein named
Cas9. The guide RNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-
binding and a user-defined “spacer”, or “targeting” sequence of about 20 nucleotides long.
Applications of the CRISPR system:
- Disabling, or knock-out of, a particular gene: After Cas-9 cuts the DNA, the cell would try to repair
the break. The more efficient the repair pathway in the cell is, the more it is prone to errors and
would most likely result in a loss-of-function mutation in the gene of interest. As CRISPR modifies
both copies of the gene at the same time, generation of knock-out animals and cell lines for gene
function studies has never been more efficient. Moreover, multiple genes can be targeted in one
manipulation, making this technique an extraordinarily powerful tool for studying complex genetic
traits or diseases that involve many genes.
- Introducing precise modifications to the target DNA: If a desired DNA sequence is provided together
with the CRISPR/Cas-9 system, it can be used by another repair pathway as a template to reconstruct
the disrupted gene sequence. The desired changes stay permanently and are also transmitted to
future generations.
- Modifications to the Cas9 enzyme have extended the application of CRISPR to selectively turn ON
and OFF target genes, or fine-tune their expression without permanently altering the gene sequence.
Since its discovery, CRISPR technology has been used extensively in animal research to engineer
disease-resistant livestock; bring back extinct species; introduce deleterious genes into malaria-
carrying mosquitoes; and modify pig genome to make pig’s organs suitable for transplant into
human. CRISPR has also been employed to create “custom designed” pets such as mini-pigs with
customized coat patterns, colorful koi fish and dogs with certain desirable traits. The CRISPR zoo is
growing rapidly but so are the ethical concerns and fears of possible ecological disasters. In human,
while CRISPR is proven to be a powerful tool to study various diseases, it is deemed not yet ready for
clinical applications. Modification of human germlines to alter genetic heritage of future generations
may also lead to unwanted consequences and is prohibited by most countries.
PRONUCLEAIRE MICRO-INJECTIE
Creation of a transgenic mouse begins with clone DNA containing the gene of interest, gene X. This
foreign DNA is injected in one of the two pronuclei of a newly fertilized mouse egg. Since the
frequency of non-homologous recombination is high, the injected DNA is randomly integrated into a
chromosome of the diploid zygote (= transgene X). Injected eggs are transferred to a foster mother
where they grow and differentiate. The resulting progenies are screened for the presence of
transgene X by PCR analysis of DNA samples. 10-30 % of the progenies will contain the transgene in
all tissues. The transgenic mouse ‘strain’ can now been analyzed for the phenotypic effects of
transgene X. Commonly assayed phenotypes are the ability to live to adulthood, the presence of
development defects and the morphology of internal tissues.
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