SUMMARY Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a counter-measure, numerous phages are known that produce proteins to block the function of Class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used Class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific “anti-CRISPRs” present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We generated a murine model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence. Using quantitative proteomics, we identified significantly increased expression of proteins involved in innate immune signaling, particularly the heterodimeric S100a8/S100a9 proteins in purified erythroblasts. S100a8 expression was significantly increased in erythroblasts, monocytes and macrophages and recombinant S100a8 was sufficient to induce an erythroid differentiation defect in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression. Our data link Rps14 haploinsufficiency to activation of the innate immune system via induction of S100A8/A9 and the p53-dependant erythroid differentiation defect in del(5q) MDS.

Parkinson's disease-causing LRRK2 mutations lead to varying degrees of Rab GTPase hyperphosphorylation. Puzzlingly, LRRK2 GTPase-inactivating mutations--which do not affect intrinsic kinase activity--lead to higher levels of cellular Rab phosphorylation than kinase-activating mutations. Here, we investigated whether mutation-dependent differences in LRRK2 cellular localization could explain this discrepancy. We discovered that blocking endosomal maturation leads to the rapid formation of mutant LRRK2+ endosomes on which LRRK2 phosphorylates substrate Rabs. LRRK2+ endosomes are maintained through positive feedback, which mutually reinforces membrane localization of LRRK2 and phosphorylated Rab substrates. Furthermore, across a panel of mutants, cells expressing GTPase-inactivating mutants formed strikingly more LRRK2+ endosomes than cells expressing kinase-activating mutants, resulting in higher total cellular levels of phosphorylated Rabs. Our study suggests that an increased probability of LRRK2 GTPase-inactivating mutants to be retained on intracellular membranes over the kinase-activating mutants leads to higher substrate phosphorylation.

Mechanisms that prevent accidental degradation of healthy mitochondria by the Pink1/Parkin mitophagy pathway are poorly understood. On the surface of damaged mitochondria, Pink1 accumulates and acts as the input signal to a positive feedback loop of Parkin recruitment, which in turn promotes mitochondrial degradation via mitophagy. However, Pink1 also transiently associates with healthy mitochondria where it could errantly recruit Parkin and thereby activate this positive feedback loop. Here, we mapped the relationship between Pink1 input levels and Parkin recruitment dynamics using quantitative live-cell microscopy and mathematical modeling. We found that Parkin is recruited to the mitochondria only if Pink1 levels exceed a threshold and only after a delay that is inversely proportional to Pink1 levels. The properties of threshold and delay emerge from the Pink1/Parkin circuit topology and provide a mechanism for cells to assess damage signals before committing to mitophagy.