Target RNA for CRISPR
The CRISPR DNA editing tool has revolutionized genetics. He helped scientists identify genes that play a key role in the development of various pathological conditions (sickle cell anemia, metastasis of lung cancer, and others). The method is limited by the fact that it is able to edit only DNA. But for many parts of the human genome, targeting DNA may be ineffective, and some other organisms, in particular RNA viruses (coronavirus, influenza virus, etc.), cannot be changed at all.
Researchers from the Neville Sanjan laboratory at the New York Genome Center and New York University, using the Cas13 enzyme, have developed an optimized platform for massive parallel genetic screening tests at the RNA level in human cells. This screening technology can be used to understand many aspects of RNA regulation and to determine the function of non-coding RNAs.
Focusing on thousands of different sites in human RNA transcripts, the researchers developed a predictive model based on machine learning to speed up the identification of the most effective guide RNAs. The new technology is available to researchers on the website and in an open source toolkit for predicting the effectiveness of RNA for custom RNA targets. It provides pre-designed guide RNAs for all human protein coding genes.
The authors intend to conduct a deep systematic analysis in order to develop key modeling principles for the most effective use of CRISPR-based RNA screening.
Cas13 are type VI CRISPR enzymes that have recently been identified as programmable RNA-controlled and RNA-targeted proteins with nuclease activity that allows knockdown of the target gene without changing the genome. This property makes Cas13 a potentially significant therapeutic agent for influencing gene expression without constantly changing the genome sequence.
The researchers also developed a set of new tools based on Cas13 and screened transcripts and permutations in mammalian cells. In total, they collected information on more than 24,000 targets for RNA editing.
In the course of this complex work, some interesting biological ideas have been discovered that can expand the use of RNA-targeted Cas13 enzymes. For example, the findings of the group contain information about which regions of the guide RNA are more important for recognizing the target RNA. Using thousands of guide RNAs with one, two, or three one-letter mismatches to their target RNA, they identified a critical "initial" region that is sensitive to mismatches between CRISPR and the target. This discovery will help scientists avoid inappropriate activity when editing RNA. Since a typical human cell expresses approximately 100,000 RNAs, the precise search of Cas13 for only the intended target is vital for screening and therapeutic applications. In addition to understanding the non-targeted action of Cas13, the information obtained can form the basis of a new generation of biosensors that can more accurately distinguish closely related types of RNA.
The use of the optimized Cas13 screening system for the detection of non-coding RNAs will significantly expand the CRISPR suite of tools for direct genetic and transcriptomic screening. The researchers found a difference in protein knockdown when targeting different coding and non-coding elements of matrix RNAs and found evidence that Cas13 competes with other RNA-binding proteins involved in transcript processing and splicing.
Article H-H.Wessels et al. Massively parallel Cas13 screens reveal principles for guide RNA design is published in the journal Nature Biotechnology.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of the New York Genome Center: New genetic screening platform using CRISPR technology for targeting thousands of genes in a massively-parallel fashion.