Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase–Cas1 fusion protein

CRISPR-Cas captures invading RNA The CRISPR (clustered regularly interspaced short palindromic repeat) system provides bacteria with an adaptive immune response. DNA captured from viruses and plasmids by CRISPR-associated protein 1 (Cas1) is used by bacteria to target the invaders' for destruction. Silas et al. discover that certain classes of the Cas1 gene are fused to a reverse transcriptase gene (RT-Cas1) (see the Perspective by Sontheimer and Marraffini). These RT-Cas1 proteins are able to capture and directly incorporate both DNA and RNA into CRISPR loci. RT-Cas1 systems could be effective against parasitic RNA species, or even to modulate bacterial gene expression. Science, this issue p. 10.1126/science.aad4234; see also p. 920 A reverse transcriptase activity captures and introduces RNA directly into CRISPR loci. INTRODUCTION Cells use a variety of mechanisms to prevent the propagation of parasitic information. A family of adaptive immune systems associated with CRISPRs in prokaryotes has been shown to protect cell populations from “selfish” DNA, including viruses and plasmids. CRISPR-mediated immunity begins with an “adaptation” phase, involving the heritable acquisition of short sequence segments (spacers) from the genome of the infectious agent by the host. This information is stored within CRISPR arrays in the host genome and is used by CRISPR-associated (Cas) nucleases in the subsequent “interference” phase to identify and disrupt infections by the same invader. CRISPR-Cas systems include those capable of interfering with DNA and RNA targets. In several characterized systems, adaptation involves acquisition from DNA templates through the action of a subset of the Cas proteins. One of these proteins (Cas1) plays a catalytic role in spacer acquisition from DNA in all systems analyzed so far. RATIONALE We sought to determine whether some CRISPR-Cas systems build CRISPR arrays through the acquisition of spacer sequences from RNA. CRISPR systems are phylogenetically grouped into five types (types I to V); in some type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). This suggests the possibility of a concerted 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 invaders. To test this hypothesis, we 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 To examine the acquisition capabilities of the MMB-1 type III-B system, we overexpressed RT-Cas1 and associated adaptation genes from MMB-1 in the native host. The resulting strains acquired a variety of new spacer elements in their type III-B CRISPR arrays. These sequences matched segments from the MMB-1 genome and our expression plasmids, with substantially more acquisitions deriving from highly transcribed genes. The transcription-associated acquisition of spacers was dependent on functional Cas1 and RT domains of the RT-Cas1 protein, supporting the idea of an RNA capture mechanism that combines Cas1 integrase activity with reverse transcription of cellular RNA. While Cas1 catalytic mutations abolished spacer acquisition, deletion or mutational inactivation of the RT domain yielded a system capable of integration with no transcriptional bias, revealing an alternative Cas1 activity on DNA substrates. To test whether the MMB-1 system can acquire sequences from RNA, we engineered a self-splicing intron into plasmid copies of two MMB-1 genes that were well sampled by RT-Cas1, simultaneously introducing mutations flanking the splice sites to yield a novel exon-junction sequence that was present as RNA but not DNA. Newly acquired spacers containing the exon-junction sequences confirmed that RT-Cas1 can acquire spacers from RNA. To investigate the relationship between the integrase and RT activities of RT-Cas1, we studied the acquisition machinery in vitro. RT-Cas1 and the associated Cas2 protein promote the precise integration of single-stranded RNA, single-stranded DNA, and double-stranded DNA oligonucleotides directly into a linear CRISPR DNA substrate, indicating that RT-Cas1 acquires spacers directly from RNA. The in vitro studies are consistent with a mechanism in which the Cas1-fused RT domain then reverse-transcribes the integrated RNA, converting it to a cDNA sequence between CRISPR repeats. The concerted integrase-RT mechanism suggested by the in vitro studies has similarities to the genomic integration mechanism used by the bacterial retrotransposons known as mobile group II introns, which encode a related RT. CONCLUSION We showed that a natural RT-Cas1 fusion protein in a type III CRISPR system can 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. RNA spacer acquisition could also contribute to immune responses against highly transcribed regions of DNA-based invaders through targeted interference at both the DNA and RNA levels. Spacer acquisition from RNA. (Left) Small segments of invasive DNA are assimilated into CRISPR arrays by Cas1 and Cas2 in a canonical spacer acquisition process that allows adaptive immunity in a wide variety of bacteria and archaea. (Right) In some type III CRISPR systems, a RT fused to Cas1 enables the acquisition of spacer sequences directly from RNA. This process might mediate adaptive immunity against RNA-based parasites. CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new 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). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.