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

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

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9

Embryos as Patients? Medical Provider Duties in the Age of CRISPR/Cas9

The CRISPR/Cas9 genome engineering platform is the first method of gene editing that could potentially be used to treat genetic disorders in human embryos. No past therapies, genetic or otherwise, have been intended or used to treat disorders in existent embryos. Past procedures performed on embryos have exclusively involved creation and implantation (e.g., in-vitro fertilization) or screening and selection of already-healthy embryos (e.g., preimplantation genetic diagnosis). A CRISPR/Cas9 treatment would evade medical malpractice law due to the early stage of the intervention and the fact that it is not a treatment for the mother. In most jurisdictions, medical professionals owe no duty to pre-viable fetuses or embryos as such, but will be held liable for negligent treatment of the mother if the treatment causes injury to a born-alive child. This issue brief discusses the science of CRISPR/Cas9, the background legal status of human embryos, and the case for considering genetically engineered embryos as patients for purposes of medical malpractice law

BATCH-GE : batch analysis of next-generation sequencing data for genome editing assessment

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome

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