In Silico Approaches for Prediction of Anti-CRISPR Proteins

Numerous viruses infecting bacteria and archaea encode CRISPR-Cas system inhibitors, known as anti-CRISPR proteins (Acr). The Acrs typically are highly specific for particular CRISPR variants, resulting in remarkable sequence and structural diversity and complicating accurate prediction and identification of Acrs. In addition to their intrinsic interest for understanding the coevolution of defense and counter-defense systems in prokaryotes, Acrs could be natural, potent on–off switches for CRISPR-based biotechnological tools, so their discovery, characterization and application are of major importance. Here we discuss the computational approaches for Acr prediction. Due to the enormous diversity and likely multiple origins of the Acrs, sequence similarity searches are of limited use. However, multiple features of protein and gene organization have been successfully harnessed to this end including small protein size and distinct amino acid compositions of the Acrs, association of acr genes in virus genomes with genes encoding helix-turn-helix proteins that regulate Acr expression (Acr-associated proteins, Aca), and presence of self-targeting CRISPR spacers in bacterial and archaeal genomes containing Acr-encoding proviruses. Productive approaches for Acr prediction also involve genome comparison of closely related viruses, of which one is resistant and the other one is sensitive to a particular CRISPR variant, and “guilt by association” whereby genes adjacent to a homolog of a known Aca are identified as candidate Acrs. The distinctive features of Acrs are employed for Acr prediction both by developing dedicated search algorithms and through machine learning. New approaches will be needed to identify novel types of Acrs that are likely to exist.     

Applications of Anti-CRISPR Proteins in Genome Editing and Biotechnology

In the ten years since the discovery of the first anti-CRISPR (Acr) proteins, the number of validated Acrs has expanded rapidly, as has our understanding of the diverse mechanisms they employ to suppress natural CRISPR-Cas immunity. Many, though not all, function via direct, specific interaction with Cas protein effectors. The abilities of Acr proteins to modulate the activities and properties of CRISPR-Cas effectors have been exploited for an ever-increasing spectrum of biotechnological uses, most of which involve the establishment of control over genome editing systems. This control can be used to minimize off-target editing, restrict editing based on spatial, temporal, or conditional cues, limit the spread of gene drive systems, and select for genome-edited bacteriophages. Anti-CRISPRs have also been developed to overcome bacterial immunity, facilitate viral vector production, control synthetic gene circuits, and other purposes. The impressive and ever-growing diversity of Acr inhibitory mechanisms will continue to allow the tailored applications of Acrs.     

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

The yeast transporter Acr3p is a low affinity As(III)/H⁺ and Sb(III)/H⁺ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane.     

Acr3p is a plasma membrane antiporter that catalyzes As(III)/H(+) and Sb(III)/H(+) exchange in Saccharomyces cerevisiae

Resistance to arsenical compounds in Saccharomyces cerevisiae as well as in a growing number of prokaryotes and eukaryotes is mediated by members of the Acr3 family of transporters. In yeast cells, it has been clearly shown that Acr3p is localized to the plasma membrane and facilitates efflux of trivalent arsenic and antimony. However, until now, the energy dependence and kinetic properties of Acr3 proteins remained uncharacterized. In this work, we show that arsenite and antimonite uptake into everted membrane vesicles via the yeast Acr3 transporter is coupled to the electrochemical potential gradient of protons generated by the plasma membrane H(+)-translocating P-type ATPase. These results strongly indicate that Acr3p acts as a metalloid/H(+) antiporter. Two differential kinetic assays revealed that Acr3p-mediated arsenite/H(+) and antimonite/H(+) exchange demonstrates Michaelis-Menten-type saturation kinetics characterized by a maximum flux for permeating metalloids. The approximate K(m) values for arsenite and antimonite transport were the same, suggesting that Acr3p exhibits similar low affinity for both metalloids. Nevertheless, the maximal velocity of the transport at saturation concentrations of metalloids was approximately 3 times higher for arsenite than for antimonite. These findings may explain a predominant role of Acr3p in conferring arsenite tolerance in S. cerevisiae.     

Discovery of potent and versatile CRISPR–Cas9 inhibitors engineered for chemically controllable genome editing

Anti-CRISPR (Acr) proteins are encoded by many mobile genetic elements (MGEs) such as phages and plasmids to combat CRISPR–Cas adaptive immune systems employed by prokaryotes, which provide powerful tools for CRISPR–Cas-based applications. Here, we discovered nine distinct type II-A anti-CRISPR (AcrIIA24–32) families from Streptococcus MGEs and found that most Acrs can potently inhibit type II-A Cas9 orthologs from Streptococcus (SpyCas9, St1Cas9 or St3Cas9) in bacterial and human cells. Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 are able to block Cas9 binding to DNA, while AcrIIA24 abrogates DNA cleavage by Cas9. Notably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, exhibiting unique anti-CRISPR characteristics. Importantly, we developed several chemically inducible anti-CRISPR variants based on AcrIIA25.1 and AcrIIA32.1 by comprising hybrids of Acr protein and the 4-hydroxytamoxifen-responsive intein, which enabled post-translational control of CRISPR–Cas9-mediated genome editing in human cells. Taken together, our work expands the diversity of type II-A anti-CRISPR families and the toolbox of Acr proteins for the chemically inducible control of Cas9-based applications.     

Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23

Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.     

Anti-CRISPR Discovery: Using Magnets to Find Needles in Haystacks

CRISPR-Cas immune systems in bacteria and archaea protect against viral infection, which has spurred viruses to develop dedicated inhibitors of these systems called anti-CRISPRs (Acrs). Like most host-virus arms races, many diverse examples of these immune and counter-immune proteins are encoded by the genomes of bacteria, archaea, and their viruses. For the case of Acrs, it is almost certain that just a small minority of nature’s true diversity has been described. In this review, I discuss the various approaches used to identify these Acrs and speculate on the future for Acr discovery. Because Acrs can determine infection outcomes in nature and regulate CRISPR-Cas activities in applied settings, they have a dual importance to both host-virus conflicts and emerging biotechnologies. Thus, revealing the largely hidden world of Acrs should provide important lessons in microbiology that have the potential to ripple far beyond the field.     

Differential roles of syntaxin 7 and syntaxin 8 in endosomal trafficking

To understand molecular mechanisms that regulate the intricate and dynamic organization of the endosomal compartment, it is important to establish the morphology, molecular composition, and functions of the different organelles involved in endosomal trafficking. Syntaxins and vesicle-associated membrane protein (VAMP) families, also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), have been implicated in mediating membrane fusion and may play a role in determining the specificity of vesicular trafficking. Although several SNAREs, including VAMP3/cellubrevin, VAMP8/endobrevin, syntaxin 13, and syntaxin 7, have been localized to the endosomal membranes, their precise localization, biochemical interactions, and function remain unclear. Furthermore, little is known about SNAREs involved in lysosomal trafficking. So far, only one SNARE, VAMP7, has been localized to late endosomes (LEs), where it is proposed to mediate trafficking of epidermal growth factor receptor to LEs and lysosomes. Here we characterize the localization and function of two additional endosomal syntaxins, syntaxins 7 and 8, and propose that they mediate distinct steps of endosomal protein trafficking. Both syntaxins are found in SNARE complexes that are dissociated by alpha-soluble NSF attachment protein and NSF. Syntaxin 7 is mainly localized to vacuolar early endosomes (EEs) and may be involved in protein trafficking from the plasma membrane to the EE as well as in homotypic fusion of endocytic organelles. In contrast, syntaxin 8 is likely to function in clathrin-independent vesicular transport and membrane fusion events necessary for protein transport from EEs to LEs.     

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.     

Estrogen activates raf-1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells

We have demonstrated that in normal and b/b rat red blood cells (RBCs) hsp70-like protein (heat shock protein 70-like) is localized in the cytosol and it is exported via exosomes during in vivo reticulocytes maturation. As we have presumed, in the mutant (b/b) rat, hsp70-like protein transfers from cytosol to the RBC membrane. In the normal rat RBCs this happens when those cells are submitted to heat stress conditions. Our study indicates that the presence of hsp70-like protein in the b/b rat RBC plasma membrane is consistent with a primary defect and is not a consequence of life long stress, i.e. hypoxia.     

Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.     

Rsp5-dependent endocytosis and degradation of the arsenite transporter Acr3 requires its N-terminal acidic tail as an endocytic sorting signal and arrestin-related ubiquitin-ligase adaptors

The yeast plasma membrane transporter Acr3 mediates efflux of toxic arsenite and antimonite. Here, we investigated the mechanisms of Acr3 turnover. We found that after arrival and residence at the plasma membrane, Acr3 is subjected to internalization followed by proteolysis in the vacuole. Endocytic degradation of Acr3 is promoted by the ubiquitin ligase Rsp5 and requires polyubiquitination of Acr3 at multiple lysine residues via lysine 63-linked ubiquitin chains. The turnover of Acr3 also depends on two arrestin-related proteins, Art3/Aly2 and Art4/Rod1, that enable recruitment of Rsp5 to its targets. Finally, we found that a short acidic patch located in the N-terminal tail of Acr3 is needed for its ubiquitination and internalization. We propose that this motif serves as an endocytic signal that facilitates binding of the arrestin-Rsp5 complexes to the Acr3 cargo.     

SEC18/NSF-independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II-coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.     

Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.     

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered,light-switchable anti-CRISPR protein

Optogenetic control of CRISPR–Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.     

Fluorescence microscopy colocalization of lipid–nucleic acid nanoparticles with wildtype and mutant Rab5–GFP: A platform for investigating early endosomal events

Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome–NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5–GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5–GFP, we found that at early time points (t < 1 h), only a fraction (≈ 35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP's membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5–Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5–GDP to the plasma membrane and not indicative of NP escape from EEs. Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5–Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes.     

Members of the Arabidopsis dynamin-like gene family,ADL1, are essential for plant cytokinesis and polarized cell growth

Polarized membrane trafficking during plant cytokinesis and cell expansion are critical for plant morphogenesis, yet very little is known about the molecular mechanisms that guide this process. Dynamin and dynamin-related proteins are large GTP binding proteins that are involved in membrane trafficking. Here, we show that two functionally redundant members of the Arabidopsis dynamin-related protein family, ADL1A and ADL1E, are essential for polar cell expansion and cell plate biogenesis. adl1A-2 adl1E-1 double mutants show defects in cell plate assembly, cell wall formation, and plasma membrane recycling. Using a functional green fluorescent protein fusion protein, we show that the distribution of ADL1A is dynamic and that the protein is localized asymmetrically to the plasma membrane of newly formed and mature root cells. We propose that ADL1-mediated membrane recycling is essential for plasma membrane formation and maintenance in plants.     

Comparative distribution of short dystrophin superfamily products in various guinea pig spermatozoa domains

In this study, the presence and cellular distribution of dystrophin family products (i.e. Dp71d, Dp71f-like protein and dystrobrevin) was examined by indirect immunofluorescence and Western blotting in guinea pig spermatozoa. Two dystrophin-associated proteins, beta-dystroglycan and alpha-syntrophin, and nNOS a protein frequently associated with alpha-syntrophin, were determined. In spermatozoa lacking plasma membrane and acrosome, Dp71f-like protein was found in the postacrosomal perinuclear theca and also in the middle piece of the flagellum. In the flagellum, Dp71f-like protein is localized together with alpha-syntrophin and nNOS. Dp71d was present in the plasma membrane of the middle piece with beta-dystroglycan, alpha-syntrophin and nNOS. Dp71d was also present in plasma membrane of the post acrosomal region, but only with nNOS. Finally, dystrobrevin was located all along skeletal flagellum structures and in the subacrosomal hemisphere of the perinuclear theca. This distinct and complementary distribution in various domains of spermatozoa may reveal a specific function for each short dystrophin family product, in the stabilization of the domains where they are located.     

B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex

B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain. The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum. Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations. One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling. Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane. Here we report the subcellular localization of B2-1 protein. Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment. B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity. Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting. Excessive overexpression of transfected B2-1 causes partial Golgi dispersion.