A Newly Discovered Bordetella Species Carries a Transcriptionally Active CRISPR-Cas with a Small Cas9 Endonuclease

Background Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. Methods The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Results Here we describe a novel Type II-C CRISPR and its associated genes—cas1, cas2, and cas9—in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Conclusions Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies

CRISPR as a Driving Force: The Model T of Biotechnology

The CRISPR system for gene editing can break, repair, and replace targeted sections of DNA. Although CRISPR gene editing has important therapeutic potential, it raises several ethical concerns. Some bioethicists worry CRISPR is a prelude to a dystopian future, while others maintain it should not be feared because it is analogous to past biotechnologies. In the scientific literature, CRISPR is often discussed as a revolutionary technology. In this paper we unpack the framing of CRISPR as a revolutionary technology and contrast it with framing it as a value-threatening biotechnology or business-as-usual. By drawing on a comparison between CRISPR and the Ford Model T, we argue CRISPR is revolutionary as a product, process, and as a force for social change. This characterization of CRISPR offers important conceptual clarity to the existing debates surrounding CRISPR. In particular, conceptualizing CRISPR as a revolutionary technology structures regulatory goals with respect to this new technology. Revolutionary technologies have characteristic patterns of implementation, entrenchment, and social impact. As such, early identification of technologies as revolutionary may help construct more nuanced and effective ethical frameworks for public policy

How the other half lives: CRISPR-Cas's influence on bacteriophages

CRISPR-Cas is a genetic adaptive immune system unique to prokaryotic cells used to combat phage and plasmid threats. The host cell adapts by incorporating DNA sequences from invading phages or plasmids into its CRISPR locus as spacers. These spacers are expressed as mobile surveillance RNAs that direct CRISPR-associated (Cas) proteins to protect against subsequent attack by the same phages or plasmids. The threat from mobile genetic elements inevitably shapes the CRISPR loci of archaea and bacteria, and simultaneously the CRISPR-Cas immune system drives evolution of these invaders. Here we highlight our recent work, as well as that of others, that seeks to understand phage mechanisms of CRISPR-Cas evasion and conditions for population coexistence of phages with CRISPR-protected prokaryotes.Comment: 24 pages, 8 figure

Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells

Characterization of CRISPR-Cas Systems in Serratia marcescens Isolated from Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

The CRISPR-Cas adaptive immune system has been attracting increasing scientific interest for biological functions and biotechnological applications. Data on the Serratia marcescens system are scarce. Here, we report a comprehensive characterisation of CRISPR-Cas systems identified in S. marcescens strains isolated as secondary symbionts of Rhynchophorus ferrugineus, also known as Red Palm Weevil (RPW), one of the most invasive pests of major cultivated palms. Whole genome sequencing was performed on four strains (S1, S5, S8, and S13), which were isolated from the reproductive apparatus of RPWs. Subtypes I-F and I-E were harboured by S5 and S8, respectively. No CRISPR-Cas system was detected in Si or S13. Two CRISPR arrays (4 and 51 spacers) were detected in S5 and three arrays (11, 31, and 30 spacers) were detected in S8. The CRISPR-Cas systems were located in the genomic region spanning from ybhR to phnP, as if this were the only region where CRISPR-Cas loci were acquired. This was confirmed by analyzing the S. marcescens complete genomes available in the NCBI database. This region defines a genomic hotspot for horizontally acquired genes and/or CRISPR-Cas systems. This study also supplies the first identification of subtype I-E in S. marcescens

Hot and crispy : CRISPR-Cas systems in the hyperthermophile Sulfolobus solfataricus

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR-Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also discuss the differences in terms of mechanisms between the subtype III-B systems in S. solfataricus and Pyrococcus furiosus.PostprintPeer reviewe

Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections.

The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein⁻Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections