Microbiology
CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion
CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader–repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3′ end of the leader.Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions
The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼27 small, fragmented rRNA genes having lengths of 22–195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol.A CRISPR/Cas9 approach reveals that the polymerase activity of DNA polymerase β is dispensable for HIV-1 infection in dividing and nondividing cells
Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase β (Pol β). However, whether human Pol β is essential for lentivirus replication in human cells is unclear. Here, we abolished DNA polymerase β (Pol β) expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate the role of Pol β in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol β–knock-out was confirmed by enhanced sensitivity to methyl methanesulfonate-induced DNA damage.CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor
Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP3R (inositol 1,4,5-trisphosphate receptor).
