Cas9 size makes it inefficient
Its large size means that Cas9 can lack efficiency when used for gene therapy. So, a large multi-institutional team worked to develop a smaller Cas enzyme that is just as active, but more efficient. The researchers selected an enzyme called AsCas12f, from the bacteria Axidibacillus sulfuroxidans. The advantage of this enzyme is that it is one of the most compact Cas enzymes found to date and less than one-third the size of Cas9. However, in previous tests it showed barely any genome activity in human cells.
“Using a screening method called deep mutational scanning, we assembled a library of potential new candidates by substituting each amino acid residue of AsCas12f with all 20 types of amino acids on which all life is based. From this, we identified over 200 mutations that enhanced genome-editing activity,” explained Prof. Nureki. “Based on insights gained from the structural analysis of AsCas12f, we selected and combined these enhanced-activity amino acid mutations to create a modified AsCas12f. This engineered AsCas12f has more than 10 times the genome-editing activity compared to the usual AsCas12f type and is comparable to Cas9, while maintaining a much smaller size.”
Animal trials with AsCas12f
The team has already carried out animal trials with the engineered AsCas12f system, partnering it with other genes and administering it to live mice. Administering treatments directly into the body is preferable to extracting cells, editing them in a lab and reinserting them into patients, which is more time-intensive and costly. The success of the tests showed that engineered AsCas12f has the potential to be used for human gene therapies, such as treating haemophilia, a disease in which the blood does not clot normally.
The team discovered numerous potentially effective combi-nations for engineering an improved AsCas12f gene-editing system, so the researchers acknowledge the possibility that the selected mutations may not have been the most optimal of all the available mixes. As a next step, computational modelling or machine learning could be used to sift through the combinations and predict which one might offer even better improvements.
“Elevating AsCas12f to exhibit genome-editing activity comparable to that of Cas9 is a significant achievement and serves as a substantial step in the development of new, more compact genome-editing tools,” said Prof. Nureki. “For us the crucial aspect of gene therapy is its potential to genuinely help patients. Using the engineered AsCas12f we developed, our next challenge is to actually administer gene therapy to aid people suffering from genetic disorders.”