Cas13 is an outlier in the CRISPR world because it targets RNA, not DNA. Once it is activated by a ssRNA sequence having complementarity to its crRNA spacer, it unleashes a nonspecific RNase activity and destroys all nearby RNA regardless of their sequence.
Recent advances have led to the development of the CRISPR-Cas13 system, which knocks down messenger RNA. Since RNA codes for the sequence of nucleic acids produced, editing the sequence of RNA can therefore temporarily edit gene expression without the serious risks linked with permanent changes to the genome.
- It could be used in the treatment of acute diseases and temporary reduction in inflammation during organ transplantation.
- The CRISPR-cas13 system can also give insights into RNA processing in disease, for example RNA editing and alternative splicing.
- Since the changes to gene levels are only transient, this allows scientists to investigate the possible gene knockouts which could cure disease. The RNA levels can be returned to normal once the CRISPR-cas13 system is removed from the cell. Due to this the changes would not be passed onto the offsprings. Some diseases are also caused due to abnormally high levels of RNA from a specific gene. Therefore, this system could be used to treat these diseases without permanently affecting the genome which could lead to disastrous side effects.
It has been shown that one member of the cas 13 family, CRISPR-Cas13a, can be used to reduce cancer-associated gene expression. The researchers also managed to design a fluorescently tagged version of this endonuclease that does not cleave the target RNA, but instead binds and allows localization analysis.
One of the major future challenges for the use of CRISPR-cas13 technology as a treatment option is suitable method of delivery into human tissues. Additionally, major ethical issues involved in editing the DNA, including worries over genome editing and genetic increase in embryos. However, this technology could enable a new and revolutionary approach for both treating and curing genetic diseases.
However, CRISPR/Cas13 has not been used for functional studies of lncRNAs or mRNAs. Furthermore, it has not been used for stable cell lines. Stable cell lines would be highly desirable in a number of reverse-genetics strategies, such as high-throughput screenings and long-term survival assays. In this, each cell stably expresses a specific gRNA that upon induction of Cas13 also integrated into the genome can induce knockdown of the target transcript. Thus, each cell is barcoded by a unique gRNA sequence. The effect of the knockdown on cellular viability could then be judged by depletion or enrichment of the specific gRNA barcodes in the cellular population.
Cas13 functions similarly to Cas9, using a nearly 64-nt guide RNA to encode target specificity. The Cas13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity is encoded by a 28 – 30-nt spacer that is complementary to the target region.