Thursday, 1 December 2016

CRISPR - The Genomic Revolution

by Kendall Field-Pellow



CRISP-what?! CRISPR is an ancient immune system component of prokaryotes such as bacteria and archaea. It is a molecular system that uses genetic material and proteins to protect a cell from infection.

The bacterium may be infected by a bacteriophage virus (type of virus which infects only bacteria, also called a phage), which means that the virus injects its genetic material (DNA or RNA which contains the code for synthesising components of a new virus particle) into a host bacterium cell. This genetic material is converted into DNA which can be integrated into the cell's genome, allowing it to be transcripted and transcribed into proteins which then form new viruses which spread to infect more hosts, and the cycle continues.

However, some bacteria survive the first infection, which means that these bacteria possess the genetic information from the phage (virus). This genetic information is inserted into the genome of the bacteria in a special location; the CRISPR loci section. CRISPR is an acronym for “Clustered Regularly Interspaced Short Palindromic Repeats”. It is a section of DNA called a repeat that has regularly spaced sequences of bases which are identical and palindromic (a symmetrical sequence such that it reads the same when spelled backwards, such as; ATCTGGTCTA), but in between these segments are different sequences which are sections of virus DNA called a spacer. This acts as an archive for DNA from previous virus attacks. When the virus re-infects the bacterium, the CRISPR Associated protein (a nuclease enzyme which is called the Cas protein coded for by the neighbouring Cas genes) forms a complex with the ‘template’ virus RNA from the CRISPR locus (called crRNA). This is complementary to the genetic material injected by the virus and so forms a complementary pair. The Cas protein then cuts the virus’s genetic material into pieces making it completely useless, protecting the cell from the virus.



Scientists now use this system for genetic engineering as it is a very effective method of gene editing. The CRISPR-Cas9 technique uses a Cas protein which is designed to have one molecule of RNA (called guide RNA or gRNA) instead of two. In bacteria there are the CRISPR RNA (crRNA) which identifies the genetic sequence which is to be cut or changed, and the tracer RNA (tracrRNA) which holds the crRNA in place with hydrogen bonds between a few complementary base pairs, but in Cas9, the single gRNA holds itself in place by forming a loop and hydrogen bonding to its own complementary base sequence between a few base pairs in a similar way to tRNA. CRISPR-Cas9 can be used to edit genes by inserting a molecule of template DNA with a specific sequence as gRNA which is designed to be complementary to a specific section of a gene, causing it to target and cut the genome in a precise location. The cell’s repair process is to attempt to reattach the two snipped ends together, however in doing so some of the bases are removed and other bases are added, which alters the base sequence; causing a mutation in the gene. Since mutations often cause the protein which the gene coded for to be non-functional, scientists can explore the effect of the absence of this protein and hence determine the effect of the gene. Other uses include transfection. This is where ‘host DNA’ is injected into the cell alongside the CRISPR-Cas9 and when the gene is cut, the host DNA is added in between, allowing scientists to study the effects of inserting a sequence in the middle of a gene and even allows genes to be added into the organism's genome.

This system is now currently used globally and is transforming the field of genetics due to its “jaw dropping simplicity and efficiency”. It can be used in cells of all organisms, not just prokaryotes, and can be used to alter any sequence, not just virus DNA. In 2015, live test rats and mice with around 99% of cells infected with HIV were injected with a treatment of specifically designed CRISPR-Cas9 which removed the HIV from over 50% of cell - By destroying the viruses genetic information in the organism's genome)!



Gene therapy is the process of changing or removing sections of genetic information which code for non-functional proteins or disorders. The CRISPR-Cas9 system has the potential to edit genes so that genetic disorders (such as Parkinson's or cystic fibrosis which have detrimental lifelong effects) are simply altered or removed from the sequence and so the person with the once permanent and irreversible disorder is now cured.

Chinese scientists have very recently used this technique to edit the genome of embryos, however due to ethical issues, the embryos were terminated before the developmental stage. This proves that CRISPR-Cas9 is the stepping stone between dreams and reality for geneticists. Lots of people have concerns that using this, or similar, method will lead to people editing the DNA of offspring before they are born so that they are not only free of inherited genetic disorders, but also possess other genetic traits such as a strengthened immune system, enhanced metabolism, perfect eyesight and endless other traits. If such a future arises, would it be more ethical to edit the genome of all babies so they they have the best possible start in life, and unethical to not genetically engineer babies as they will be at a disadvantage to those whom have edited genomes. This is a very complex ethical issue, but by making our voices heard and our opinions known, we can ensure that both scientists and the general population reach an agreeable conclusion regarding what actions should be allowed and which should be prohibited. I believe that peaceful negotiations are a far more beneficial than just shutting down research programs because said programs could make life changing advancements and if they are shut down, we are not engaging correctly with the scientific community. Also a slightly more fearful thought is that if we do not collaborate to reach an agreeable ethical solution regarding gene editing, this development will only continue in places such as those which have a dubious ethical record and a tendency to be aggressive which could use the technology to make terrible and immoral moves like, hypothetically, an army of modified ‘super soldiers’!

The CRISPR-Cas9 technique is considerably easier, cheaper and more accurate in comparison to current methods of gene editing. This is very exciting because it is allowing the field of genetic engineering to advance rapidly faster than the rate that other fields usually progress!

However, we are far from creating designer babies, but such an outcome may be on the horizon for the near future. Although the use of CRISPR-Cas9 is effective and more accurate, it has its drawbacks, including:

     The Cas nuclease enzyme can cut sequences in the wrong place
     The Cas nuclease enzyme can cut different sequences which are quite similar, causing the wrong gene to be changed
     The Cas nuclease enzyme can insert new genes the wrong way around, causing them to be translated incorrectly and a completely different (and therefore non-fictional) protein to be produced
     The Cas nuclease enzyme can delete whole sections
     The gene editing process is not exact and so the mutation could cause or even worsen a genetic disorder
     It is difficult to get the CRISPR-Cas9 system into in vivo somatic (body) cells compared to in vitro (in the lab). Often a modified virus is used, but this would cause the subject to be infected with a virus which could have unpredictable side effects

     Without editing embryos, it is impossible to make heritable changes which will be passed on to offspring which means that any corrections to the genome to reverse genetic disorders in the parent will not be passed on to offspring and so the offspring is at risk of inheriting the genetic disease.

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