Cas4 Nucleases Define the PAM, Length, and Orientation of DNA Fragments Integrated at CRISPR Loci.

To achieve adaptive and heritable immunity against viruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide in vivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.

Type III-A CRISPR systems as a versatile gene knockdown technology.

CRISPR-Cas systems are functionally diverse prokaryotic antiviral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis, and S. thermophilus) were each expressed on a single plasmid in E. coli, and the efficiency and specificity of gene knockdown was assessed by northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and noncoding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

Hyper-stimulation of Pyrococcus furiosus CRISPR DNA uptake by a self-transmissible plasmid.

Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation.

Genome-wide identification of zero nucleotide recursive splicing in Drosophila.

Recursive splicing is a process in which large introns are removed in multiple steps by re-splicing at ratchet points--5' splice sites recreated after splicing. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing. Here we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila. Finally, we identify four recursively spliced human genes, one of which is also recursively spliced in Drosophila. Together, these results indicate that recursive splicing is commonly used in Drosophila, occurs in humans, and provides insight into the mechanisms by which some large introns are removed.

Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus.

To acquire CRISPR-Cas immunity against invasive mobile genetic elements, prokaryotes must first integrate fragments of foreign DNA into their genomic CRISPR arrays for use in future invader silencing. Here, we found that the hyperthermophilic archaeaon, Pyrococcus furiosus, actively incorporates DNA fragments (spacers) from both plasmid (foreign) and host genome (self) sequences into its seven CRISPR loci. The majority of new spacers were derived from DNA immediately downstream from a 5'-CCN-3' protospacer adjacent motif (PAM) that is critical for invader targeting. Interestingly, spacers were preferentially acquired from genome or plasmid regions corresponding to active transposons, CRISPR loci, ribosomal RNA genes, rolling circle origins of replication, and areas where plasmids recombined with the host chromosome. A common feature of the highly sampled spacers is that they arise from DNA regions expected to undergo DNA nicking and/or double-strand breaks. Taken together with recent results from bacterial systems, our findings indicate that free DNA termini and PAMs are conserved features important for CRISPR spacer uptake in diverse prokaryotes and CRISPR-Cas systems. Moreover, lethal self-targeting by CRISPR systems may contribute to host genome stability by eliminating cells undergoing active transposon mobility or chromosomal uptake of autonomously replicating foreign mobile genetic elements.

A role for A-to-I RNA editing in temperature adaptation.

A-to-I RNA editing can recode mRNAs, giving organisms the option to express diverse, functionally distinct protein isoforms. Here, we propose that RNA editing is inherently geared for temperature adaptation because it tends to recode to smaller, less stabilizing amino acids. Studies on how editing affects protein function support this idea.

Histones direct site-specific CRISPR spacer acquisition in model archaeon.

CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.

Pruning and Tending Immune Memories: Spacer Dynamics in the CRISPR Array.

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) is a type of prokaryotic immune system that is unique in its ability to provide sequence-specific adaptive protection, which can be updated in response to new threats. CRISPR-Cas does this by storing fragments of DNA from invading genetic elements in an array interspersed with short repeats. The CRISPR array can be continuously updated through integration of new DNA fragments (termed spacers) at one end, but over time existing spacers become obsolete. To optimize immunity, spacer uptake, residency, and loss must be regulated. This mini-review summarizes what is known about how spacers are organized, maintained, and lost from CRISPR arrays.

Complete Genome Sequence of Streptococcus ratti JH145.

We report here the complete genome sequence of Streptococcus ratti strain JH145. Streptococcus ratti is a cariogenic species of mutans streptococcus that has been isolated from rat and human teeth. The strain JH145, derived from strain BHT-2, is interesting for oral health because it does not produce cariogenic lactic acid but shows robust biofilm production.

Complete Genome Sequence of Industrial Dairy Strain Streptococcus thermophilus DGCC 7710.

We report here the complete genome sequence of Streptococcus thermophilus DGCC 7710. S. thermophilus is widely used in industrial dairy production.

The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing.

RNA editing by adenosine deamination alters genetic information from the genomic blueprint. When it recodes mRNAs, it gives organisms the option to express diverse, functionally distinct, protein isoforms. All eumetazoans, from cnidarians to humans, express RNA editing enzymes. However, transcriptome-wide screens have only uncovered about 25 transcripts harboring conserved recoding RNA editing sites in mammals and several hundred recoding sites in Drosophila. These studies on few established models have led to the general assumption that recoding by RNA editing is extremely rare. Here we employ a novel bioinformatic approach with extensive validation to show that the squid Doryteuthis pealeii recodes proteins by RNA editing to an unprecedented extent. We identify 57,108 recoding sites in the nervous system, affecting the majority of the proteins studied. Recoding is tissue-dependent, and enriched in genes with neuronal and cytoskeletal functions, suggesting it plays an important role in brain physiology.