An anti-CRISPR that pulls apart a CRISPR-Cas complex.

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.

American Dog Ticks (Dermacentor variabilis) as Biological Indicators of an Association between the Enteric Bacterium Moellerella wisconsensis and Striped Skunks (Mephitis mephitis) in Southwestern Manitoba, Canada.

Total genomic (g)DNA from 100 American dog ticks (Dermacentor variabilis) collected from humans, dogs, raccoons, and skunks near Minnedosa (Manitoba, Canada) in 2005 was tested for the presence of Moellerella wisconsensis (Gammaproteobacteria: Enterobacteriales) using PCR. Although two gDNA samples derived from ticks attached to two striped skunks (Mephitis mephitis) contained M. wisconsensis DNA, it is unlikely that D. variabilis is a vector of this bacterium. Genomic DNA prepared from the washes of the external surfaces of these two ticks (i.e., before DNA extraction from the whole tick) and another two ticks attached to same skunks were also PCR positive for M. wisconsensis. This suggests that ticks acquired the bacterium by physical contact with contaminated or infected skunks. However, it does not exclude the possibility that the ticks may have also imbibed the bacterium from their host blood and lymph. Nonetheless, the results of this molecular study suggest that the four adult D. variabilis represent biological indicators of the presence of M. wisconsensis in association with their vertebrate hosts (i.e., striped skunks). Additional work is needed to determine if M. wisconsensis is present in the blood and lymph of striped skunks in southwestern Manitoba and if there are potential health risks for persons coming into contact with infected animals.

Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.

Three genetically distinct clades of Anaplasma phagocytophilum in Ixodes scapularis.

Human granulocytic anaplasmosis (HGA) is an emerging disease in Canada because of range expansion by the arthropod vector, Ixodes scapularis. These ticks carry the Ap-ha variant of Anaplasma phagocytophilum (Ap-ha), which has been implicated in causing HGA, and the Ap-variant 1, which is not associated with human infection. We report the detection of 13 genotypes of the ankyrin (ankA) gene among 76 infected blacklegged ticks. Haplotype network and phylogenetic analyses revealed that the ankA genotypes corresponding to the Ap-ha variant did not form a monophyletic assemblage. They formed two distinct clades (Clades I and III), one of which was genetically more similar in nucleotide and amino acid sequences to genotypes of Ap-variant 1 that comprised Clade II. Additional work is needed to explore the evolutionary history of A. phagocytophilum in North America, and to determine if there are differences in pathogenicity or clinical symptoms associated with the two divergent groups of the Ap-ha variant given the significant differences in ankA amino acid sequence.