Crispr / Cas9 is the molecular shear that has revolutionized biology in recent years. But it is still not perfect and numerous researches are underway to make it as precise as possible. The discovery of an “advanced” version of the system, evoCas9, which proved almost impeccable in the tests, is entirely Italian. An evolutionary approach was used to achieve this.
What is Crispr / Cas9 and how does it work?
Let’s go over it together! First of all, it is a recent technology, developed in 2012, and it was not just our merit: we were inspired by a component of the immune system of bacteria, which they use to defend themselves against viruses. Cas proteins “copy and paste” a part of the genome of the virus into the Crispr locus of the bacterial chromosome, so as to keep it in memory and quickly neutralize it. The system synthesizes an exact copy of the genetic fragment of the virus previously incorporated, but in RNA format, which is associated with a protein called Cas9, an enzyme that cuts DNA: the RNA strand guides the Cas9 protein exactly to the virus, which succumbs under the blows of the relentless scissors.
The researchers optimized the system to modify DNA with an accuracy unthinkable until a few years ago. The Cas9 protein is associated with an RNA strand that functions as a “navigator” and directs it to the point of the genome that is to be modified; Cas9 cuts and the cell repairs DNA using the complementary filament as a mold (we know that DNA is a double helix and that even during replication each of the two filaments acts as a mold for the synthesis of the other, thanks to the complementarity of the nitrogen bases ). By doing so, we can replace individual “letters” in DNA, for example by correcting mutations that lead to disease, or simply by turning off harmful genes.
Towards an increasingly precise range … and made-in-Italy!
The premises are good, but it is still early to think about its regular therapeutic use. Crispr / Cas9 is a precision instrument, but even the best ones can make mistakes and one of its great limitations is the risk of non-specific activity. Unfortunately, in this case, the error is not allowed, since cutting the genome “haphazardly” could be extremely dangerous for the patient.
Researchers from the University of Trento have further perfected the system, minimizing non-specific activity. His performance was brilliant, even in the long term. How did they do it?
There were two possible strategies: improving the RNA “navigator” or optimizing the Cas9 “scissors”. They opted for the second, following an evolutionary approach … in a test tube! The method is called “direct evolution” and is used to evolve proteins towards a predefined purpose. Numerous variants of the Cas9 protein were first generated and then assembled into Streptococcus pyogenes cells. The scientists then put up a screening system to visually recognize the most specific variants and selected only the bacteria that had incorporated them for a subsequent round of mutagenesis, screening and selection and so on. They then mimicked what happens in nature, but they put their hand in it, masterfully directing the evolution towards the desired purpose.
The more specific variants were then selected and the corresponding mutations identified. The best four were combined into a single protein, generating evoCas9, a real “scalpel” that stood out in all tests for accuracy and specificity. Thanks to evoCas9, we are ever closer to working on DNA with the precision of a surgeon.
Source: Casini, A., et al. (2018). A highly specific SpCas9 variant is identified by in vivo
screening in yeast. Nature Biotechnol.