CRISPR-Cas RNA Targeting Using Transient Cas13a Expression in Nicotiana benthamiana
Scientists from Kansas State University described a new method for CRISPR-Cas RNA targeting using transient Cas13a expression in Nicotiana benthamiana. The details are published in RNA Abundance Analysis and discussed in the book Methods in Molecular Biology.
The use of CRISPR-Cas prokaryotic immune system for single-stranded RNA targeting is expected to have vital importance in RNA analysis and engineering. The class 2 Type VI CRISPR-Cas13 system is an RNA-guided RNA-nuclease system that can bind and cleave target single-stranded RNA substrates in a sequence-specific manner. Aside from RNAi, the Cas13a system has applications from manipulating RNA modifications, including editing RNA sequence and use as a nucleic acid detection tool.
The new protocol presented uses Cas13a ortholog from Leptotrichia buccalis for transient expression in plant cells providing antiviral defense. The vital details are presented in the book chapter, including information on cloning the Cas13 protein, crRNA guide cassette, performing transient Agrobacterium-mediated expression of the necessary Cas13a components, and target RNA-virus, visualization of virus infection, and molecular quantification of viral accumulation using quantitative PCR.