Foreign Genetic Elements Research Articles

Viral Anti-CRISPR Tactics: No Success without Sacrifice

Co-evolution with their bacterial hosts has led to viral countermeasures against CRISPR-mediated immunity. In a recent issue of Cell, Landsberger etal. (2018) and Borges etal. (2018) report that cooperation among bacteriophages and multiple infection events are necessary to overcome CRISPR immune responses.

Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro.

Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides, some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however, they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome, specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors, including thermostable single-stranded DNA binding proteins and UvrD-like helicase, in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates, including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.

Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA

Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling.

On the Origin and Evolutionary Relationships of the Reverse Transcriptases Associated With Type III CRISPR-Cas Systems.

Reverse transcriptases (RTs) closely related to those encoded by group II introns but lacking the intron RNA structure have been found associated with type III clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, a prokaryotic immune system against invading viruses and foreign genetic elements. Two models have been proposed to explain the origin and evolutionary relationships of these RTs: (i) the “single point of origin” model, according to which these RTs originated from a single acquisition event in bacterial, with the various protein domains (RT, RT-Cas1, and Cas6-RT-Cas1 fusions) corresponding to single points in evolution; and (ii) the “various origins” model, according to which, independent acquisition events in different evolutionary episodes led to these fusions. We tested these alternative hypotheses, by analyzing and integrating published datasets of RT sequences associated with CRISPR-Cas systems and inferring phylogenetic trees by maximum likelihood (ML) methods. The RTs studied could be grouped into 13 clades, mostly in bacteria, in which they probably evolved. The various clades appear to form three independent lineages in bacteria and a recent lineage in archaea. Our data show that the Cas6 domain was acquired twice, independently, through RT-Cas1 fusion, in the bacterial lineages. Taken together, there more evidence to support the “various origins” hypothesis.

Argonaute Facilitates the Lateral Diffusion of the Guide along Its Target and Prevents the Guide from Being Pushed Away by the Ribosome.

Argonaute (AGO) proteins play central roles in nucleic acid-guided interference that regulates gene expression and defend against foreign genetic elements in all life. Although much progress has been made with respect to the function of argonaute proteins in target recognition and cleavage, the detailed mechanism of their biological functions is not fully understood. Here, using atomic force microscopy-based single-molecule force spectroscopy, we studied target-guide dissociation in the absence or presence of Thermus thermophilus AGO (TtAGO). Our results indicated that AGO changed the fundamental properties of target-guide interaction. Dissociation of the target from the guide is easier in the lateral direction of the nucleic acid in the presence of AGO protein but harder in the longitudinal direction. Our results support the idea that one-dimensional diffusion of the RNA-induced silencing complex (RISC) along the target strand is more efficient than three-dimensional diffusion and explain the priority of RISC binding over the ribosome complex during translation elongation.

Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches.

Adaptive immunity against foreign genetic elements conferred by the CRISPR systems in microbial species has been repurposed as a revolutionary technology for wide-ranging biological applications-chiefly genome engineering. Biochemical, structural, genetic, and genomics studies have revealed important insights into their function and mechanisms, but most ensemble studies cannot observe structural changes of these molecules during their function and are often blind to key reaction intermediates. Here, we review the use of single molecule approaches such as fluorescent particle tracking, FRET, magnetic tweezers, and atomic force microscopy imaging in improving our understanding of the CRISPR toolbox.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

ABSTRACTCRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.

Diversity and microevolution of CRISPR loci in Helicobacter cinaedi.

Helicobacter cinaedi is associated with nosocomial infections. The CRISPR-Cas system provides adaptive immunity against foreign genetic elements. We investigated the CRISPR-Cas system in H. cinaedi to assess the potential of the CRISPR-based microevolution of H. cinaedi strains. A genotyping method based on CRISPR spacer organization was carried out using 42 H. cinaedi strains. The results of sequence analysis showed that the H. cinaedi strains used in this study had two CRISPR loci (CRISPR1 and CRISPR2). The lengths of the consensus direct repeat sequences in CRISPR1 and CRISPR2 were both 36 bp-long, and 224 spacers were found in the 42 H. cinaedi strains. Analysis of the organization and sequence similarity of the spacers of the H. cinaedi strains showed that CRISPR arrays could be divided into 7 different genotypes. Each genotype had a different ancestral spacer, and spacer acquisition/deletion events occurred while isolates were spreading. Spacer polymorphisms of conserved arrays across the strains were instrumental for differentiating closely-related strains collected from the same hospital. MLST had little variability, while the CRISPR sequences showed remarkable diversity. Our data revealed the structural features of H. cinaedi CRISPR loci for the first time. CRISPR sequences constitute a valuable basis for genotyping, provide insights into the divergence and relatedness between closely-related strains, and reflect the microevolutionary process of H. cinaedi.

Genome sequences of lower Great Lakes Microcystis sp. reveal strain-specific genes that are present and expressed in western Lake Erie blooms.

Blooms of the potentially toxic cyanobacterium Microcystis are increasing worldwide. In the Laurentian Great Lakes they pose major socioeconomic, ecological, and human health threats, particularly in western Lake Erie. However, the interpretation of “omics” data is constrained by the highly variable genome of Microcystis and the small number of reference genome sequences from strains isolated from the Great Lakes. To address this, we sequenced two Microcystis isolates from Lake Erie (Microcystis aeruginosa LE3 and M. wesenbergii LE013-01) and one from upstream Lake St. Clair (M. cf aeruginosa LSC13-02), and compared these data to the genomes of seventeen Microcystis spp. from across the globe as well as one metagenome and seven metatranscriptomes from a 2014 Lake Erie Microcystis bloom. For the publically available strains analyzed, the core genome is ~1900 genes, representing ~11% of total genes in the pan-genome and ~45% of each strain’s genome. The flexible genome content was related to Microcystis subclades defined by phylogenetic analysis of both housekeeping genes and total core genes. To our knowledge this is the first evidence that the flexible genome is linked to the core genome of the Microcystis species complex. The majority of strain-specific genes were present and expressed in bloom communities in Lake Erie. Roughly 8% of these genes from the lower Great Lakes are involved in genome plasticity (rapid gain, loss, or rearrangement of genes) and resistance to foreign genetic elements (such as CRISPR-Cas systems). Intriguingly, strain-specific genes from Microcystis cultured from around the world were also present and expressed in the Lake Erie blooms, suggesting that the Microcystis pangenome is truly global. The presence and expression of flexible genes, including strain-specific genes, suggests that strain-level genomic diversity may be important in maintaining Microcystis abundance during bloom events.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

Investigating a rare methicillin-resistant Staphylococcus aureus strain: first description of genome sequencing and molecular characterization of CC15-MRSA.

PurposeMethicillin resistant Staphylococcus aureus CC15 strains (CC15-MRSA) have only been sporadically described in literature. This study was carried out to describe the genetic make-up for this rare MRSA strain.MethodsFour CC15-MRSA isolates collected in Riyadh, Saudi Arabia, between 2013 and 2014 were studied. Two isolates were from clinical infection and 2 from retail meat products. Whole genome sequencing was carried out using Illumina HiSeq2500 genome analyzer.ResultsAll the CC15-MRSA isolates had the multilocus sequence typing profile ST1535, 13–13-1–1-81-11-13, which is a single locus variant of ST15. Of the 6 contigs related to the SCC element, one comprised a recombinase gene ccrAA, ccrC-PM1, fusC and a helicase, another one included mvaS, dru, mecA and 1 had yobV and Q4LAG7. The SCC element had 5 transposase genes, namely 3 identical paralogs of tnpIS431 and 2 identical paralogs of tnpIS256. Two identical copies of a tnpIS256-based insertion element flank the aacA-aphD gene. Two copies of this insertion element were present with 1 located in the SCC element and another inserted into the sasC gene. A short 3 kb region, which lacks any bacteriophage structural genes and site-specific DNA integrase, was inserted into the hlb gene. The hsdM and the 5’-part of the hsdS gene are replaced by a copy of the hsdM/hsdS paralogs from νSaβ giving rise to a new chimeric paralog of hsdS in νSaα.ConclusionCC15-MRSA shows a novel SCCmecV/SCCfus composite element. Its variant of hsdM/hsdS probably facilitated uptake of foreign mobile genetic elements that promoted emergence of CC15-MRSA. Close surveillance is needed to monitor spread and emergence of further CC15 MRSA strains.

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.

CRISPR Editing Technology in Biological and Biomedical Investigation.

The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness, and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy, and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future. J. Cell. Biochem. 118: 3586-3594, 2017. © 2017 Wiley Periodicals, Inc.

In silico genomic insights into aspects of food safety and defense mechanisms of a potentially probiotic Lactobacillus pentosus MP-10 isolated from brines of naturally fermented Aloreña green table olives.

Lactobacillus pentosus MP-10, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. The genome sequence of L. pentosus MP-10 is currently considered the largest genome among lactobacilli, highlighting the microorganism’s ecological flexibility and adaptability. Here, we analyzed the complete genome sequence for the presence of acquired antibiotic resistance and virulence determinants to understand their defense mechanisms and explore its putative safety in food. The annotated genome sequence revealed evidence of diverse mobile genetic elements, such as prophages, transposases and transposons involved in their adaptation to brine-associated niches. In-silico analysis of L. pentosus MP-10 genome sequence identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) as an immune system against foreign genetic elements, which consisted of six arrays (4–12 repeats) and eleven predicted cas genes [CRISPR1 and CRISPR2 consisted of 3 (Type II-C) and 8 (Type I) genes] with high similarity to L. pentosus KCA1. Bioinformatic analyses revealed L. pentosus MP-10 to be absent of acquired antibiotic resistance genes, and most resistance genes were related to efflux mechanisms; no virulence determinants were found in the genome. This suggests that L. pentosus MP-10 could be considered safe and with high-adaptation potential, which could facilitate its application as a starter culture and probiotic in food preparations.

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleasesto defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvCpocket. These structural insights, validated bybiochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.

Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage.

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation—resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system–type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

Recent advances in sequencing methods have prompted an upsurge in research into the modification of diseaseassociated genes or genes involved in drug resistance. In the early days of genome-editing, immunogenic and difficult to deliver tools with high off-target effects such as Zinc Finger Nucleases (ZFNs) and Transcription activatorlike effector nucleases (TALENs) proteins were used. This was followed by the discovery of the Clustered Regularly Interspaced Short Palindromic Repeat s, CRISPR/Cas9 system. This “self-non-self-discriminatory” natural defence mechanism in bacteria and archaea identifies and degrades extrachromosomal genomes or foreign genetic elements to prevent them from integrating into the prokaryotic genomes. Unlike traditional gene therapies that can merely insert a gene in a random pattern, the inexpensive and expedient CRISPR/ Cas9 system was harnessed by scientists to precisely cut, add or manipulate multiple DNA sequences at specific sites. This review will focus on the most recent studies in genome-editing by giving an overview of the past and modern methods in this field with an emphasis on the bacterial adaptive immune system called CRISPR/Cas9. This article will also analyze the applications of CRISPR/ Cas9 in rewriting the human genome in clinical and research settings and its potential future therapeutic applications. In addition, we shall consider the technical complexities which need to be overcome for the safe and effective delivery of this novel therapy and how the ethical concerns associated with this revolutionary genome engineering technique have constrained research in germ-line genome-editing. Furthermore, we shall highlight the role of scientific regulatory committees in evaluating and assessing safety issues such as potential complications before translating this proof-of-concept evidential approach to clinical reality. More research is required to explore the risk of hazardous genome edits and to fine-tune and improve this novel therapeutic system. Although the inexpensive and easy to use CRISPR/ Cas9 platform paves new ways for precise genome editing, clinical application is still at an early stage.

CRISPR-Cas systems as weapons against pathogenic bacteria

CRISPR-Cas systems (Clustered Regularly Interspaced Short Palindromic Repeats) are the adaptive immune system of bacteria and archaea. They target foreign genetic elements thanks to small RNAs able to guide Cas nucleases to destroy them. These nucleases can be reprogrammed to target chromosomal sequences rather than invasive genetic elements. Whereas targeting the genome of eukaryotic cells enables the efficient genesis of mutations, DNA breaks induced by Cas nucleases are lethal in bacteria. This property can be used in the development of novel antimicrobial strategies. CRISPR-Cas systems can be delivered to target bacteria using bacteriophage capsids in order to specifically eliminate bacteria carrying antibiotic resistance genes or virulence factors. These technologies enable the development of novel tools based on CRISPR-Cas systems to specifically eliminate pathogenic bacteria and precisely modify the composition of various microbiomes.

Overview of CRISPR-Cas9 Biology.

Prokaryotes use diverse strategies to improve fitness in the face of different environmental threats and stresses, including those posed by mobile genetic elements (e.g., bacteriophages and plasmids). To defend against these elements, many bacteria and archaea use elegant, RNA-directed, nucleic acid-targeting adaptive restriction machineries called CRISPR -: Cas (CRISPR-associated) systems. While providing an effective defense against foreign genetic elements, these systems have also been observed to play critical roles in regulating bacterial physiology during environmental stress. Increasingly, CRISPR-Cas systems, in particular the Type II systems containing the Cas9 endonuclease, have been exploited for their ability to bind desired nucleic acid sequences, as well as direct sequence-specific cleavage of their targets. Cas9-mediated genome engineering is transcending biological research as a versatile and portable platform for manipulating genetic content in myriad systems. Here, we present a systematic overview of CRISPR-Cas history and biology, highlighting the revolutionary tools derived from these systems, which greatly expand the molecular biologists' toolkit.

The solution structure of an anti-CRISPR protein.

Bacterial CRISPR–Cas adaptive immune systems use small guide RNAs to protect against phage infection and invasion by foreign genetic elements. We previously demonstrated that a group of Pseudomonas aeruginosa phages encode anti-CRISPR proteins that inactivate the type I-F and I-E CRISPR–Cas systems using distinct mechanisms. Here, we present the three-dimensional structure of an anti-CRISPR protein and map a functional surface that is critical for its potent inhibitory activity. The interaction of the anti-CRISPR protein with the CRISPR–Cas complex through this functional surface is proposed to prevent the binding of target DNA.