Advanced Gene Editing Technology Enables RNA Tracking in Living Cells

Genomics researchers have used a modified version of the CRISPR/Cas9 gene editing technique to track the movement of RNA in living cells.

Defective RNA transport has been linked to a number of conditions ranging from autism to cancer, and researchers have sought methods to measure RNA movement to facilitate development of treatments for these conditions.

CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas complex comprises a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR-Cas9 system is composed of two parts: The Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides (CRISPRs) that shepherd the Cas9 protein to the target gene on a DNA strand.

CRISPR systems are phylogenetically grouped into five types (types I to V). In addition to the CRISPR/Cas9 complex, CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new viral threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT) enzyme. Such an arrangement suggested the possibility of a spacer integration mechanism involving Cas1 integrase activity and the reverse transcription of RNA to DNA.

In the current study, investigators at the University of California, San Diego (USA) demonstrated that nuclease-inactive Streptococcus pyogenes CRISPR/Cas9 could bind RNA in a nucleic-acid-programmed manner that allowed the endogenous tracking of RNA in living cells. The investigators focused on the RNA that encoded the proteins ACTB, TFRC, and CCNA2. Results published in the March 17, 2016, online edition of the journal Cell showed how a complex of Cas9 fused to a fluorescent protein marker revealed the movement of RNA into stress granules, a cluster of proteins and RNAs that form in a cell's cytosol during periods of cellular stress.

"This work is the first example, to our knowledge, of targeting RNA in living cells with CRISPR-Cas9," said senior author Dr. Gene Yeo, associate professor of cellular and molecular medicine at the University of California, San Diego. "Our current work focuses on tracking the movement of RNA inside the cell, but future developments could enable researchers to measure other RNA features or advance therapeutic approaches to correct disease-causing RNA behaviors."

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University of California, San Diego