Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups.

Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavour other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighbouring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

Approaches to study CRISPR RNA biogenesis and the key players involved.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an inheritable and adaptive immune system against phages and foreign genetic elements in many bacteria and archaea. The three stages of CRISPR-Cas immunity comprise adaptation, CRISPR RNA (crRNA) biogenesis and interference. The maturation of the pre-crRNA into mature crRNAs, short guide RNAs that target invading nucleic acids, is crucial for the functionality of CRISPR-Cas defense systems. Mature crRNAs assemble with Cas proteins into the ribonucleoprotein (RNP) effector complex and guide the Cas nucleases to the cognate foreign DNA or RNA target. Experimental approaches to characterize these crRNAs, the specific steps toward their maturation and the involved factors, include RNA-seq analyses, enzyme assays, methods such as cryo-electron microscopy, the crystallization of proteins, or UV-induced protein-RNA crosslinking coupled to mass spectrometry analysis. Complex and multiple interactions exist between CRISPR-cas-encoded specific riboendonucleases such as Cas6, Cas5d and Csf5, endonucleases with dual functions in maturation and interference such as the enzymes of the Cas12 and Cas13 families, and nucleases belonging to the cell's degradosome such as RNase E, PNPase and RNase J, both in the maturation as well as in interference. The results of these studies have yielded a picture of unprecedented diversity of sequences, enzymes and biochemical mechanisms.

Divergent methylation of CRISPR repeats and cas genes in a subtype I-D CRISPR-Cas-system.

BACKGROUND: The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality. RESULTS: The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated (5mCG6mATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5mC methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GGm4CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system. CONCLUSIONS: The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5mC methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5mC methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5CGATCG DNA-methylation marks in conjugation and DNA transformation.

A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis.

The yeast Kluyveromyces lactis has been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressible LAC4 promoter (P LAC4 ) is the most widely used promoter to drive recombinant protein expression in K. lactis However, P LAC4 is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, P LAC4 is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novel K. lactis promoter system that is both strong and tightly controllable. We first tested several different endogenous K. lactis promoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of two K. lactis promoters, namely, the strong constitutive P GAP1 promoter and the carbon source-sensitive P ICL1 promoter. We demonstrate that P350 is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350 hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins in K. lactisIMPORTANCE The yeast Kluyveromyces lactis is an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use in K. lactis P350 is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be "autoinduced" at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803.

Specialized RNA endonucleases are critical for efficient activity of the CRISPR-Cas defense mechanisms against invading DNA or RNA. Cas6-type enzymes are the RNA endonucleases in many type I and type III CRISPR-Cas systems. These enzymes are diverse and critical residues involved in the recognition and cleavage of RNA substrates are not universally conserved. Cas6 endonucleases associated with the CRISPR-Cas subtypes I-A, I-B, I-C, I-E and I-F, as well as III-B have been studied from three archaea and four bacteria thus far. However, until now information about subtype I-D specific Cas6 endonucleases has remained scarce. Here, we report the biochemical analysis of Cas6-1, which is specific for the crRNA maturation from the subtype I-D CRISPR-Cas system of Synechocystis sp. PCC 6803. Assays of turnover kinetics suggest a single turnover mechanism for Cas6-1. The mutation of conserved amino acids R29A, H32A-S33A and H51A revealed these as essential, whereas the parallel mutation of R175A-R176A led to a pronounced and the K155A mutation to a slight reduction in enzymatic activity. In contrast, the mutations R67A, R81A and K231A left the enzymatic activity unchanged. These results are in accordance with the predominant role of histidine residues in the active site and of positively charged residues in RNA binding. Nevertheless, the protein-RNA interaction site seems to differ from other known systems, since imidazole could not restore the mutated histidine site.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

CRISPR-Based Technologies for Metabolic Engineering in Cyanobacteria.

In metabolic engineering, the production of industrially relevant chemicals, via rational engineering of microorganisms, is an intensive area of research. One particular group of microorganisms that is fast becoming recognized for their commercial potential is cyanobacteria. Through the process of photosynthesis, cyanobacteria can use CO2 as a building block to synthesize carbon-based chemicals. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-dependent approaches have rapidly gained popularity for engineering cyanobacteria. Such approaches permit markerless genome editing, simultaneous manipulation of multiple genes, and transcriptional regulation of genes. The drastically shortened timescale for mutant selection and segregation is especially advantageous for cyanobacterial work. In this review, we highlight studies that have implemented CRISPR-based tools for the metabolic engineering of cyanobacteria.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation.

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements.

The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR-Cas subtype III-Bv system.

Specialized RNA endonucleases for the maturation of clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs) are critical in CRISPR-CRISPR-associated protein (Cas) defence mechanisms. The Cas6 and Cas5d enzymes are the RNA endonucleases in many class 1 CRISPR-Cas systems. In some class 2 systems, maturation and effector functions are combined within a single enzyme or maturation proceeds through the combined actions of RNase III and trans-activating CRISPR RNAs (tracrRNAs). Three separate CRISPR-Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Whereas Cas6-type enzymes act in two of these systems, the third, which is classified as subtype III-B variant (III-Bv), lacks cas6 homologues. Instead, the maturation of crRNAs proceeds through the activity of endoribonuclease E, leaving unusual 13- and 14-nucleotide-long 5'-handles. Overexpression of RNase E leads to overaccumulation and knock-down to the reduced accumulation of crRNAs in vivo, suggesting that RNase E is the limiting factor for CRISPR complex formation. Recognition by RNase E depends on a stem-loop in the CRISPR repeat, whereas base substitutions at the cleavage site trigger the appearance of secondary products, consistent with a two-step recognition and cleavage mechanism. These results suggest the adaptation of an otherwise very conserved housekeeping enzyme to accommodate new substrates and illuminate the impressive plasticity of CRISPR-Cas systems that enables them to function in particular genomic environments.

RNase Y of Staphylococcus aureus and its role in the activation of virulence genes.

RNase Y of Bacillus subtilis is a key member of the degradosome and important for bulk mRNA turnover. In contrast to B. subtilis, the RNase Y homologue (rny/cvfA) of Staphylococcus aureus is not essential for growth. Here we found that RNase Y plays a major role in virulence gene regulation. Accordingly, rny deletion mutants demonstrated impaired virulence in a murine bacteraemia model. RNase Y is important for the processing and stabilization of the immature transcript of the global virulence regulator system SaePQRS. Moreover, RNase Y is involved in the activation of virulence gene expression at the promoter level. This control is independent of both the virulence regulator agr and the saePQRS processing and may be mediated by small RNAs some of which were shown to be degraded by RNase Y. Besides this regulatory effect, mRNA levels of several operons were significantly increased in the rny mutant and the half-life of one of these operons was shown to be extremely extended. However, the half-life of many mRNA species was not significantly altered. Thus, RNase Y in S. aureus influences mRNA expression in a tightly controlled regulatory manner and is essential for coordinated activation of virulence genes.