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Some CRISPR Gene Editing Complexes Target RNA

By LabMedica International staff writers
Posted on 15 Mar 2016
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Image: Representation of the crystal structure of a CRISPR-associated protein (Photo courtesy of Wikimedia Commons).
Image: Representation of the crystal structure of a CRISPR-associated protein (Photo courtesy of Wikimedia Commons).
A team of molecular microbiologists have demonstrated that in some bacteria the CRISPR/Cas genome editing complex can edit RNA as well as DNA.

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. 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 enzyme 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. This would enable the acquisition of new spacers from RNA, potentially generating adaptive immunity against RNA-based viruses. To test this hypothesis, investigators at the Carnegie Institution for Science Department of Plant Biology (Stanford, CA, USA) characterized the spacer acquisition machinery of the RT-Cas1–containing type III-B CRISPR system in the bacterium Marinomonas mediterranea (MMB-1), by means of in vivo assays and in vitro reconstitution.

Results published in the February 26, 2016, issue of the journal Science revealed that a natural RT-Cas1 fusion protein in a type III CRISPR system could enable the acquisition of new spacers directly from RNA. With other type III CRISPR systems known to target RNA for degradation, RT-associated CRISPR-Cas systems would effectively generate adaptive immunity against RNA parasites.

Contributing author Dr. Devaki Bhaya professor of biology at the Carnegie Institution of Science Department of Plant Biology, said, "The team has demonstrated that this biochemical process can occur in the lab, and based on this information, the CRISPR/Cas system may confer immunity against RNA-based invaders out there in the wild. It is gratifying to see how much we can learn from the extraordinary protein diversity that exists in the microbial and viral world, especially when it is combined with rigorous biochemistry."

Related Links:

Carnegie Institution for Science Department of Plant Biology


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