CRISPR Cas9 and genetic editing on human embryonic cells

In recent days, numerous scientific publications – STAT, Scientific American of Nature and the sciences – have announced the perfectly successful modification of human embryonic cells, thanks to CRISPR Cas9 technology, the so-called “cut and paste” of DNA. The experiment was conducted by Shoukhrat Mitalipov and other researchers from Oregon Health & Science University and the Salk Institute for Biological Studies in La Jolla1. In fact, it is not the first time in the world. Compared to previous studies, the difference lies in the improvement of the technique developed by the American team, to avoid two main harmful consequences: mosaicism and the onset of new mutations.

CRISPR Cas 9 has an almost thirty-year history
The system was first discovered in Japan by Ishino Y et all back in 1987 and then published in a scientific journal. However, they did not understand the potential of the technique well. It was taken up and studied for genomic editing – separately – by two researchers: the American Jennifer Doudna and the French Emmanuelle Charpentier (still contending the patent today); but the years of consecration are only recent:

2013, the technique was among “the 10 most important discoveries of 2013” according to Science;
2015, human embryos were modified for the first time
2017, in China, was used to modify DNA of normal cells.
To date, it has numerous applications in different areas.

The name CRISPR Cas9 is composed of “CRISPR”, which stands for “Clustered Regularly Interspaced Short Palindromic Repeats” and “Cas”, instead, for “CRISPR-Associated Systems” 3 proteins.
It is an RNA-enzyme complex, belonging to the immune system4 of microorganisms. It has the function of affecting and eliminating the foreign genetic elements of viruses or plasmids, integrated in the host / infected bacterial genome. It can be found in both bacteria and Archaea4.5.

The entry of foreign DNA activates a defense mechanism in the microorganism that leads to the transcription and translation of Cas nucleases, enzymes capable of attacking and fragmenting it. Subsequently, the pieces of DNA are introduced into the “CRISPR locus”, ie a region composed of a series of sequences and empty spaces, in which it is inserted. In addition, next to the locus, we find the leader sequence and the genes for Cas.

In truth, the immune system of microorganisms has developed three ways to combat invasion and as a result, there are many Cas proteins. What we are interested in is Cas9, the only protein required in type II, that is, the strategy adopted by genetic editing.
In general, all systems have in common the generation of the crRNA strands (cr = CRISPR – RNA), called biogenesis.

In the case of II, it will be helped by a tracrRNA (a trans activating crRNA) 6 to mature and ultimately train the guide to our Cas9 (sgRNA = strand guide RNA), which will be transported inside. Once assembled, the system will recognize and match the DNA corresponding to the guide RNA, making a cut at the site. The void will then be filled through the classic natural mechanisms.

Technically speaking, it is a fairly simple method, at an affordable cost. In this case, it has been used to treat hypertrophic heart disease2 and the results have been incredible, without the onset of further genetic errors.

In the future, it can be applied for the treatment of numerous genetic diseases dependent on a single gene (about 10,000). For this reason, it is necessary to refine the protocol more and more so that it can be used in clinical practice, as stated by the scientists themselves.

Despite the benefits, it opens a bioethical debate of no less importance: how far can genetic engineering go? 1,2

We just have to wait for the next developments!

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