Over the last decade, ∼20−30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs— short interfering RNAs (siRNAs) and microRNAs (miRNAs)—act in both somatic and germline line-ages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment.

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria. C lustered, regularly interspaced, short palindromic repeat (CRISPR) loci are present in~40% of eubacterial genomes and nearly all archaeal genomes sequenced to date and consist of short (~24 to 48 nucleotides) repeats separated by similarly sized unique spacers (1, 2). They are generally flanked by a set of CRISPR-associated (cas) protein-coding genes (3-5). The CRISPR spacers and repeats are transcribed and processed into small CRISPR RNAs (crRNAs) (4, 6-8) that specify acquired immunity against bacteriophage infection by a mechanism that relies on the strict identity between CRISPR spacers and phage targets (3, 4).The rise of hospital-and communityacquired methicillin-and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA, respectively) is directly linked to the horizontal transfer of antibiotic resistance genes by plasmid conjugation (9, 10). S. aureus and S. epidermidis strains are the most common causes of nosocomial infections (11-13), and conjugative plasmids can spread from one species to the other. Although the S. epidermidis strain American Type Culture Collection (ATCC) 12228 (14) lacks CRISPR sequences, the clinically isolated strain RP62a (15) contains a CRISPR locus (Fig. 1A and fig. S1A) that includes a spacer (spc1) that is homologous to a region of the nickase (nes) gene found in all sequenced staphylococcal conjugative plasmids ( fig. S1B), including those from MRSA and VRSA strains (9,16,17).To test whether spc1 prevents plasmid conjugation into S. epidermidis RP62a, we disrupted the sequence match by introducing nine silent mutations into the nes target in the conjugative plasmid pG0400 (18), generating pG0(mut) (Fig. 1B) (19). We tested whether both wild-type and mutant pG0400 transferred from S. aureus strain RN4220 (20) into either of the two S. epidermidis strains (Fig. 1D and fig. S1C). Although the conjugation frequency of both plasmids was similar for the CRISPR-negative ATCC 12228 strain, only pG0(mut) was transferred into the CRISPR-positive RP62a strain and with a frequency similar to that of wild-type pG0400 in the control ATCC 12228 strain. These r...

The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila.

Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs -small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway.The acquisition of new genes that confer a selective advantage is an important factor in genome evolution. Considerable proportions of bacterial and archaeal genomes consist of genes derived from the exchange of genetic material among related or unrelated species 1 , which is known as horizontal gene transfer (HGT). HGT occurs by uptake of environmental DNA (transformation) or by the incorporation of heterologous DNA carried on mobile genetic elements, such as plasmids (conjugation) and bacteriophages (transduction) 2 .However, only a miniscule fraction of acquired genes confers an immediate selective advantage. Therefore bacteria and archaea have developed many mechanisms to prevent HGT, such as DNA restriction and surface exclusion 2 . Recently, arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) have been identified as determinants of a novel genetic interference pathway that limits at least two major routes of HGT -conjugation and transduction. Like eukaryotic RNA interference (RNAi) and Competing interests statementThe authors declare no competing financial interests. DATABASES NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript related pathways (with which CRISPR interference is analogous but not homologous), CRISPR interference provides the host with an efficient antiviral defence mechanism.In contrast to other gene transfer and phage defence mechanisms, CRISPR interference is an adaptive immune system that can be reprogrammed to reject invading DNA molecules that have not been previously encountered. CRISPRs are separated by short spacer sequences that match bacteriophage or plasmid sequences and specify the targets of interference. Upon phage infection, CRISPR arrays can acquire new repeat-spacer units that match the challenging phage. Cells with this extended CRISPR locus will survive phage infection and thrive. Therefore the spacer content of CRISPR arrays reflects the many different phages and plasmids that have been encountered by the host, and these spacers can be expanded rapidly in response to new invasions. Accordingly, CRISPRs constitute a 'genetic memory' that ensures the rejection of new, returning and ever-present invading DNA m...

All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway1–9. CRISPR loci are present in ~ 40% and ~90% of sequenced bacterial and archaeal genomes respectively10 and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations1, 11–13. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats1–9. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes14–16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences2, 17–19. CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive “protospacer” DNA2, 3, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, suggesting that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.