CRISPR-Cas12-based nucleic acids detection systems.

Because of the outstanding contribution in genome editing, CRISPR has undoubtedly become the most popular technology around the world and two pioneers are awarded the Nobel Prize in Chemistry this year. Besides, along with the discovery of nonspecific trans-cleavage activities of several Cas proteins such as Cas12 and Cas13, many CRISPR-based molecular diagnostic systems have been successfully created, showing advantages in sensitivity, specificity and operation convenience. Among them, systems with Cas12, which targets DNA and trans-cleaves single-stranded DNA probes, are both simple and highly efficient. Here in this review, we mainly focus on the Cas12-based methods and briefly discuss their applications in nucleic acids detection and beyond.

Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps.

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.

DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis.

Regulation of DNA phosphorothioate modifications by the transcriptional regulator DptB in Salmonella.

DNA phosphorothioate (PT) modifications, with one non-bridging phosphate oxygen replaced with sulfur, are widely but sporadically distributed in prokaryotic genomes. Short consensus sequences surround the modified linkage in each strain, although each site is only partially modified. The mechanism that maintains this low-frequency modification status is still unknown. In Salmonella enterica serovar Cerro 87, PT modification is mediated by a four-gene cluster called dptBCDE. Here, we found that deletion of dptB led to a significant increase in intracellular PT modification level. In this deletion, transcription of downstream genes was elevated during rapid cell growth. Restoration of dptB on a plasmid restored wild-type levels of expression of downstream genes and PT modification. In vitro, DptB directly protected two separate sequences within the dpt promoter region from DNase I cleavage. Each protected sequence contained a direct repeat (DR). Mutagenesis assays of the DRs demonstrated that each DR was essential for DptB binding. The observation of two shifted species by gel-shift analysis suggests dimer conformation of DptB protein. These DRs are conserved among the promoter regions of dptB homologs, suggesting that this regulatory mechanism is widespread. These findings demonstrate that PT modification is regulated at least in part at the transcriptional level.

Pathological phenotypes and in vivo†DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella.

Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification.

Different Detection Strategies of Pediocin-Like Produced by Pediococcus pentosaceus.

In this study, Pediococcus pentosaceus C-2-1 and C23221 contained genes encoding penocin and pediocin PA-1, mined by antiSMASH. The penocin structural gene pedA from Pediococcus pentosaceus C-2-1 was successfully expressed in Escherichia coli BL21. The presence of a 6.5 kDa recombinant penocin was confirmed by Tricine-SDS-PAGE, and the specific activity increased by 1.54-fold. The bacteriocins produced by Pediococcus pentosaceus C23221 were purified using acetic ether extraction, Sepharose Fast Flow, Sephadex G-25 gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC); the amino acid sequence of this bacteriocin was identical to pediocin PA-1 by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), which confirmed the expression of pediocin PA-1 gene; and the specific activity increased by 24.39-fold. The heterologous expression and purification of bacteriocins have proved the expression of pediocin-like produced by Pediococcus pentosaceus. This provides a theoretical basis for the subsequent development and application of pediocin-like.

Three functional replication origins of the linear and artificially circularized plasmid SCP1 of Streptomyces coelicolor.

Previous reports showed that the large linear plasmid SCP1 of Streptomyces coelicolor A3(2) contains a 5.4 kb centrally located replication locus. We report here that SCP1 actually contains three internal replication loci. Subcloning of the 5.4 kb sequence identified a 3.2 kb minimal locus (rep1A/repB/iteron) that determined propagation in Streptomyces lividans. The two newly identified replication genes, rep2A and rep3A, resembled the rep gene of Streptomyces circular plasmid pZL12. Transcription start points of the three replication genes were determined. The three replication loci could independently determine propagation in linear mode in S. lividans. Individual and sequential deletions of the rep1A and rep3A genes were successful. The SCP1-derived linear plasmids with deletions of the rep1A and/or rep3A genes still propagated in similar copy numbers, were inherited largely stable and were transferred efficiently by conjugation in S. coelicolor. Interestingly, SCP1 can be artificially circularized to yield a 280 kb circular plasmid, circular SCP-1 (C-SCP1), which contains the three replication loci. Strikingly, the copy numbers, inheritance and transfer of C-SCP1 resembled that of the linear SCP1 plasmids. Transcripts of the rep1A, rep2A and rep3A genes in linear or artificially circularized SCP1 were detected at all the time points of strain growth.

A putative transglycosylase encoded by SCO4132 influences morphological differentiation and actinorhodin production in Streptomyces coelicolor.

Here we report that tgdA, a novel gene encoding a putative transglycosylase, affects both the morphological differentiation and the yield of blue-pigmented compound actinorhodin in Streptomyces coelicolor. The tgdA null mutant displays sparse aerial hyphae and irregular spore chains frequently lacking chromosomal DNA. Elevated actinorhodin production coincides with the overexpression of actII-orf4 in mutant. tgdA expression is temporally and developmentally regulated. The tgdA orthologs in Streptomyces avermilitis and Streptomyces lividans also affect differentiation.

Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species.

BACKGROUND: Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. RESULTS: We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. CONCLUSIONS: This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.

[Plasmid pCQ4 and its phage phiCQ4 of endophytic Streptomyces sp. from Artemisia annua L].

OBJECTIVE: Large plasmid pCQ4 was detected in Streptomyces sp. W75 from Artemisia annua L. We clonined, sequenced, analyzed and characterized pCQ4. METHODS: Southern hybridization was used to determine restriction map of pCQ4. To clone the full-length of pCQ4, conjugation and recombinational cloning in a BAC vector were used. RESULTS: The complete nucleotide sequence of pCQ4 consisted of 84833-bp, encoding 129 ORFs which 40 ORFs resembled these of bacterial phages. W75 culture could infect W75 cured of pCQ4 and formed plaques on plate. Phage particle (phiCQ4) was observed by transmission electron microscopy. LinearphiCQ4 DNA was detected on pulsed-field gel electrophoresis. Comparison to Streptomyces plasmid-phage pZL12, genes encoding major phage structural proteins resembled that of pCQ4. CONCLUSION: Streptomyces plasmid pCQ4 could be transformed into lytic phagephiCQ4, and the phage segment on pCQ4 might be a mobile unit.

Characterization of the multiple CRISPR loci on Streptomyces linear plasmid pSHK1.

The complete nucleotide sequence including the novel telomere sequence of Streptomyces linear plasmid pSHK1 consists of 187,263-bp, 158 genes, in which 51 genes resemble those of the linear plasmid SCP1 of Streptomyces coelicolor A3(2), and 20 genes encode transposases. Strikingly, the repetitive CRISPRs (clustered regularly interspaced short palindromic repeats) and cas (CRISPR-associated) genes were found, including a cluster of eight cas genes, in the order cas2B-cas1B-cas3B-cas5-cas4-cas2A-cas1A-cas3A, bracketed by a pair of divergent CRISPRs, and five other dispersed CRISPRs. The cas2B-cas1B-cas3B-cas5 or cas4-cas2A-cas1A genes were co-transcribed. Protein-protein interactions between Cas5 and Cas1A, 2A, 2B, 3B were detected by yeast two-hybrids, indicating a critical role of Cas5 for the formation of protein complexes. By polymerase chain reaction and Southern hybridization, 12 cas4 genes including three on linear plasmids were found among 75 newly isolated Streptomyces strains. The paired-CRISPRs and bracketed cas were also conserved in several other Streptomyces or actinomycete species. However, unlike other bacteria, the CRISPRs-cas in pSHK1 could not provide immunity against introduction of phage ΦC31 and plasmid containing the particular spacers in Streptomyces.

Characterization of the replication, transfer, and plasmid/lytic phage cycle of the Streptomyces plasmid-phage pZL12.

We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

[Cloning and identification of telomere and internal origin of the endophytic Streptomyces linear plasmid pYY8L from Allium fistulosum].

UNLABELLED: Strain 36R-2-1B was isolated from Allium fistulosum and identified as Streptomyces spp., harboring an approximate 280 kb linear plasmid designated pYY8L. OBJECTIVE: Cloning, sequencing and analysis of telomere and internal replication origin of pYY8L. METHODS: pYY8L telomere was cloned by a modified procedure--"alkaline treatment and enzyme digestion in gel". The internal replication origin of pYY8L was cloned by construction of a cosmid library and then subcloning. RESULTS: An approximate 280 kb DNA band (pYY8L) of strain 36R-2-1B was detected by pulsed-field gel electrophoresis. The 152-bp telomere, containing six small palindromes and potentially forming complicated secondary structure, was cloned and sequenced. The replication origin of pYY8L was initially cloned on a cosmid and then sub-cloned as a 4891-bp autonomous replication sequence. This sequence was predicted to contain six genes, two of them resembling replication genes of Streptomyces linear plasmid SCP1, but their adjacent iterons were different. CONCLUSION: New telomere and internal replication origin of Streptomyces linear plasmid pYY8L were cloned and identified. This is the first example of report on telomere and replication genes of linear plasmid from endophytic Streptomyces.

A novel autoantibody targeting calreticulin is associated with cancer in patients with idiopathic inflammatory myopathies.

OBJECTIVES: To investigate the prevalence and clinical significance of anti-calreticulin autoantibodies (anti-CRT Ab) in a large cohort of idiopathic inflammatory myopathy (IIM) patients. METHODS: Sera from 469 patients with IIM, 196 patients with other connective tissue diseases, 28 patients with solid tumors and 81 healthy controls were screened for anti-CRT Ab by enzyme-linked immunosorbent assay using human recombinant CRT protein. Sera from 35 IIM patients were tested using an immunoprecipitation assay to confirm the presence of anti-CRT Ab. Subsequently, IIM-cancer patients were identified and divided into new-onset, remission and recurrent groups based on their cancer status. The relationships between anti-CRT Ab levels and IIM disease activity were also investigated. RESULTS: Serum anti-CRT Ab was detected positive in 81 of the 469 (17.3%) IIM patients. Immunoprecipitated bands were observed at a molecular weight of 60 kDa corresponding to the CRT protein. The IIM patients with anti-CRT Ab more frequently had cancers compared to the patients without anti-CRT Ab. Moreover, the prevalence of anti-CRT Ab differed according to the cancer status. The IIM patients with recurrent cancers had a much higher prevalence of anti-CRT Ab than those with cancers in remission. Also, serum anti-CRT Ab levels positively correlated with disease activity at baseline and at follow-up visits. CONCLUSION: We report the existence of serum anti-CRT Ab in IIM patients and demonstrate the possible association of anti-CRT Ab with malignancy in IIM patients. Serum anti-CRT Ab could serve as a novel candidate marker of cancer in IIM patients.

Erratum: Author Correction: CRISPR-Cas12a-assisted nucleic acid detection.

[This corrects the article DOI: 10.1038/s41421-018-0028-z.].

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus.

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.

Multiplex gene regulation by CRISPR-ddCpf1.

The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical studies.

A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette.

As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.

In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.

A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.