Scientists have developed a novel CRISPR system, it allows at the same time in several places to cut the DNA and remove genes. In addition to the widely known Cas9, it includes Cas12a, which has ribonuclease activity and can be cut one RNA at a few guides. System called CHyMErA, and she was more efficient than the usual CRISPR/Cas9 to turn off genes, study the properties of pairs of genes that affect each other, paralegal and alternative exons. Article published in the journal Nature Biotechnology.
CRISPR/Cas system is most known for the fact that by using them, scientists can modify the genome of organisms. However, this technology allows not only change the DNA, but also to investigate the function of genes that cannot be understood through other technologies. For example, using CRISPR to investigate the genetic interaction is a deviation from the expected phenotype, which result from overlapping of several genes.
One of the important issues — what functions are and how to affect the phenotype analogichnye genes — identical copies of DNA, which arose as a result of nutrigenomic duplications. Such events occurred in the course of evolution, and gradually copies of genes can obtain different roles. In addition, scientists are studying the functioning of alternative exons — sections that can be cut out of the mRNA, resulting in the matrix of the same gene can build different proteins.
For such studies the necessary tools with which to influence directly on different combinations of genes. There is a system in which Cas9 working with two or more RNA guides, however they have some limitations: different promoters and sites of RNA can recombine, and the necessary sequence is broken. The other nucleases, Cas12a, there is an internal ribonuclease activity, it can split hidaway RNA and make it into a set of different sequences. However, the effectiveness of Cas12a low.
Thomas Galatopoulos of Pornazzi (Thomas Gonatopoulos-Pournatzis) from the University of Toronto and his colleagues have developed a system, called CHyMErA (Cas Hybrid Editing and for Multiplexed Screening Applications, “Hybrid Cas for multiple editing and screening”). In her Cas9 and Cas12a work together and use hybrid hidaway RNA.
Scientists have created a library of hybrid Vidovich RNA that recognize various important genes of human and mouse (450 genes) and the number of exons. Then used a convolutional neural network to predict the properties and efficiency of the hybrid Vidovich RNA. The algorithm is trained on data obtained as a result of the introduction of different CHyMErA cells in mice and humans (5 thousand sequences).
Technology tried to turn off individual genes, and to the famous pairs influence each other genes by turning off one or both sequences. Then use CHyMErA off each of the 1344 paralogon of famous couples and cut out different exons.
The predictive ability of convolutional neural network was 77 percent, it was determined the active galouye RNA Cas12a better than counterparts that were used before. Off of the same gene twice with CHyMErA was significantly more effective than using Cas9 and Cas12a separately. Using the system confirmed the already known genetic interaction between homologous genes and paralogous, even managed to find a new pair of paralogos that affect each other. Also CHyMErA has been effective for cutting out alternative exons.
This year the technology of CRISPR/Cas9 for the first time used on a human for the treatment of blindness. Read more about how CRISPR works, you can read in our article “Remember those letters”.