Microbial Genetics: CRISPR Memories Of

RESEARCH HIGHLIGHTS

Nature Reviews Genetics | Published online 7 Mar 2016; doi:10.1038/nrg.2016.31

MICROBIAL GENETICS The ligation of both RNA and DNA oligonucleotides to the CRISPR substrate was abolished by a mutation in the CRISPR memories of RNA Cas1 active site, but only the ligation of RNA oligonucleotides was abolished by CRISPR–Cas systems are widespread authors focused on the type III-B deletion of the RT domain, consistent among prokaryotes and provide a system from the melanogenic marine with the RT-dependent bias towards form of adaptive immunity. Short bacterium Marinomonas mediterranea highly transcribed regions that was segments of invading DNA are captured strain MMB‑1. Overexpression of observed in vivo. and integrated as interspersed spacers RT–Cas1 in MMB-1 resulted in the Finally, using radiolabelled dNTPs within CRISPR arrays, providing a new acquisition of spacers within the to facilitate the detection of cDNA, heritable memory that directs the native CRISPR array. Interestingly, these the authors tested whether an RNA targeting of matching nucleic acid spacers were enriched for sequences oligonucleotide integrated into a CRISPR sequences for digestion. In a paper from highly transcribed genes, and array could be reverse transcribed by published in Science, Silas et al. now this enrichment was abolished by the RT–Cas1–Cas2 complex in vitro. report that a subset of CRISPR systems deletion of the RT domain. Such an The cDNA products detected were has the ability to acquire spacers not RT-dependent bias is consistent with consistent with the reverse transcription only from DNA but from RNA as well. spacer acquisition involving the of integrated RNA primed by the 3′ the acquisition Previous genomic analyses had shown reverse transcription of RNA. end of the CRISPR DNA of the opposite that, in some CRISPR systems, the The authors assessed this possibility in strand. The authors suggest that these of RNA CRISPR-associated protein Cas1, which MMB‑1 through the introduction of a cDNAs may represent an intermediate spacers by catalyses spacer integration (along with plasmid encoding genes that contained in the acquisition of a fully integrated type III CRISPR Cas2), is fused to a putative reverse self-splicing introns. The subsequent double-stranded DNA spacer. transcriptase (RT). Silas et al. carried detection of newly integrated spacers The team concludes that the systems may out phylogenetic analyses of the genes corresponding to spliced transcripts acquisition of RNA spacers by type III enable the encoding RT–Cas1 fusions, and found confirmed the ability of RT–Cas1 to CRISPR systems may enable the targeting of that all classifiable fusions belonged facilitate the acquisition of spacers targeting of parasitic RNA species, such exclusively to the type III class of from RNA. as RNA phages, and may contribute parasitic RNA CRISPR systems. To investigate the mechanism to immune responses towards highly species To explore the ability of RT–Cas1 of spacer acquisition, the authors transcribed regions of DNA phages and to facilitate spacer acquisition, the incubated purified RT–Cas1 and Cas2 plasmids via an interference system with DNA or RNA oligonucleotides, targeting DNA, RNA or both. In addition, a CRISPR DNA substrate, and the authors highlight the possibility that deoxynucleoside 5ʹ-triphosphates RNA spacer acquisition could also occur (dNTPs) to enable reverse transcription. in species that have seperately encoded These assays revealed that either RT and Cas1 proteins. DNA or RNA oligonucleotides could Denise Waldron be ligated directly to the CRISPR substrate. Moreover, the sensitivity of a ORIGINAL ARTICLE Silas, S. et al. Direct CRISPR ligated RNA oligonucleotide to RNase H spacer acquisition from RNA by a natural reverse transcriptase‑Cas1 fusion protein. Science http:// digestion demonstrated its existence as dx.doi.org/10.1126/science.aad4234 (2016) part of an RNA–DNA hybrid, consistent FURTHER READING Sontheimer, E. J. & Marraffini, with reverse transcription of the ligated L.A. CRISPR goes retro. Science 351, 920–921 PhotoDisc/ (2016) Getty Images RNA to generate cDNA.