Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks.

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells.

PUF family translational repressors are conserved developmental regulators, but the molecular function provided by the regions flanking the PUF RNA-binding domain is unknown. In C. elegans, the PUF proteins FBF-1 and FBF-2 support germline progenitor maintenance by repressing production of meiotic proteins and use distinct mechanisms to repress their target mRNAs. We identify dynein light chain DLC-1 as an important regulator of FBF-2 function. DLC-1 directly binds to FBF-2 outside of the RNA-binding domain and promotes FBF-2 localization and function. By contrast, DLC-1 does not interact with FBF-1 and does not contribute to FBF-1 activity. Surprisingly, we find that the contribution of DLC-1 to FBF-2 activity is independent of the dynein motor. Our findings suggest that PUF protein localization and activity are mediated by sequences flanking the RNA-binding domain that bind specific molecular partners. Furthermore, these results identify a new role for DLC-1 in post-transcriptional regulation of gene expression.

Improving the specificity of nucleic acid detection with endonuclease-actuated degradation.

Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.

3' UTRs are the primary regulators of gene expression in the C. elegans germline.

How genes are regulated to produce the correct assortment of proteins for every cell type is a fundamental question in biology. For many genes, regulation begins at the DNA level with the use of promoter sequences to control transcription. Regulation can also occur after transcription using sequences in the 3' untranslated region (UTR) of the mRNA to affect mRNA stability and/or translation [1]. The C. elegans gonad is an excellent tissue to study gene regulation during development: In the adult, germ cells are arranged in order of differentiation, with undifferentiated progenitors at one end of the gonad, cells in meiotic prophase in the middle, and gametes at the other end [2]. Using a transgenic assay, we have compared the contribution of promoters and 3' UTRs to gene regulation during germline development. We find that for most genes tested, 3' UTRs are sufficient for regulation. With the exception of promoters activated during spermatogenesis, promoters are permissive for expression in all germ cell types (from progenitors to oocytes and sperm). In progenitors, 3' UTRs inhibit the production of meiotic and oocyte proteins by posttranscriptional mechanisms involving PUF- and KH-domain RNA-binding proteins. Our findings indicate that many genes rely primarily on 3' UTRs, not promoters, for regulation during germline development.

Protein-based condensation mechanisms drive the assembly of RNA-rich P granules.

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In Caenorhabditis elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is a modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-MS/MS with multiple reaction monitoring.

An AccQ*Tag ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ*Tag-UPLC-ESI-MS/MS) method for fast, reproducible, and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with a photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ*Tag amino acids with multiple reaction monitoring, varied from 29 to 39 V, whereas the collision energy varied from 15 to 35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentration ranges of 3 x 10(-3)-25 pmol/muL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the range of 0.08-1.08%. The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ*Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/MS for amino acid quantitation using AccQ*Tag derivatization.

Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos.

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.

Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double-Stranded DNA Donors.

Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology-directed repair of DNA breaks induced by the Cas9 RNA-guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30-40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9-crRNA-tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors.

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes.

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2 We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures.

Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3.

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3's access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.

Copper pathways in Plasmodium falciparum infected erythrocytes indicate an efflux role for the copper P-ATPase.

Copper, like iron, is a transition metal that can generate oxygen radicals by the Fenton reaction. The Plasmodium parasite invades an erythrocyte host cell containing 20 microM copper, of which 70% is contained in the Cu/Zn SOD (cuprozinc superoxide dismutase). In the present study, we follow the copper pathways in the Plasmodium-infected erythrocyte. Metal-determination analysis shows that the total copper content of Percoll-purified trophozoite-stage-infected erythrocytes is 66% that of uninfected erythrocytes. This decrease parallels the decrease seen in Cu/Zn SOD levels in parasite-infected erythrocytes. Neocuproine, an intracellular copper chelator, arrests parasites at the ring-to-trophozoite stage transition and also specifically decreases intraparasitic levels of Cu/Zn SOD and catalase. Up to 150 microM BCS (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulphonic acid), an extracellular copper chelator, has no effect on parasite growth. We characterized a single copy PfCuP-ATPase (Plasmodium falciparum copper P-ATPase) transporter, which, like the Crypto-sporidium parvum copper P-ATPase, has a single copper-binding domain: 'Met-Xaa-Cys-Xaa-Xaa-Cys'. Recombinant expression of the N-terminal metal-binding domain reveals that the protein specifically binds reduced copper. Transcription of the PfCuP-ATPase gene is the highest at late ring stage/early trophozoite, and is down-regulated in the presence of neocuproine. Immunofluorescence and electron microscopy indicate the transporter to be both in the parasite and on the erythrocyte membrane. Both the decrease in total copper and the location of the PfCuP-ATPase gene indicate a copper-efflux pathway from the infected erythrocyte.

High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.

Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.

Identification of suppressors of mbk-2/DYRK by whole-genome sequencing.

Screening for suppressor mutations is a powerful method to isolate genes that function in a common pathway or process. Because suppressor mutations often do not have phenotypes on their own, cloning of suppressor loci can be challenging. A method combining whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) mapping (WGS/SNP mapping) was developed to identify mutations with visible phenotypes in C. elegans. We show here that WGS/SNP mapping is an efficient method to map suppressor mutations without the need for previous phenotypic characterization. Using RNA-mediated interference to test candidate loci identified by WGS/SNP mapping, we identified 10 extragenic and six intragenic suppressors of mbk-2, a DYRK family kinase required for the transition from oocyte to zygote. Remarkably, seven suppressors are mutations in cell-cycle regulators that extend the timing of the oocyte-to-zygote transition.

Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans.

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2A(PPTR-½). Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.

Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalis.

Development of resistance against metronidazole and mechanisms responsible for this process were studied in a sexually transmitted pathogen of humans, Trichomonas vaginalis. Monitoring of changes in metabolism and protein expression that accompanied increasing resistance of strains derived from a common drug-susceptible parent (TV 10-02) showed the multistep character of the process. The aerobic type of resistance known to occur in isolates from patients non-responsive to treatment appeared at the earliest stage, followed by development of the anaerobic type of resistance which was accompanied by gradual loss of hydrogenosomal proteins associated with drug-activating pathways [pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin]. Unexpectedly, the loss of PFOR did not result in acquisition of full anaerobic resistance, thus indicating an alternative source of electrons required for the drug activation. These data suggest involvement of the oxidative decarboxylation of malate in hydrogenosomes, catalysed by NAD(+)-dependent malic enzyme and subsequent transfer of reduced equivalents to the drug via NADH:ferredoxin oxidoreductase and ferredoxin. Accordingly, all components of this pathway were eliminated before the resistance was fully developed. Resistant Trichomonas vaginalis compensated the impaired function of hydrogenosomes by enhanced conversion of pyruvate to lactate in the cytosol. Further analysis of the two key enzymes involved in metronidazole activation by Northern blotting and assay for nascent mRNA showed that the insufficient expression of the PFOR protein results from decreased gene transcription, while down-regulation of malic enzyme is controlled at the mRNA level.

Incorporation of iron into Tritrichomonas foetus cell compartments reveals ferredoxin as a major iron-binding protein in hydrogenosomes.

The intracellular transport of iron and its incorporation into organelles are poorly understood processes in eukaryotes and virtually unknown in parasitic protists. The transport of iron is of particular interest in trichomonads, which possess hydrogenosomes instead of mitochondria. The metabolic functions of hydrogenosomes, which contain a specific set of FeS proteins, entirely depend on iron acquisition. In this work the incorporation of iron into the cattle parasite Tritrichomonas foetus was monitored. Iron was efficiently taken up from (59)Fe-nitrilotriacetic acid and accumulated in the cytosol (88.9 %) and hydrogenosomes (4.7 % of the total radioactivity). Using atomic absorption spectrophotometry, an unusually high steady-state iron concentration in hydrogenosomes was determined [54.4+/-1.1 nmol Fe (mg protein)(-1)]. The concentration of iron in the cytosol was 13.4+/-0.5 nmol Fe (mg protein)(-1). Qualitative analysis of incorporated iron was performed using native gradient PAGE. The majority of the (59)Fe in the cytosol appeared as the labile-iron pool, which represents weakly bound iron associated with compounds of molecular mass ranging from 5000 to 30000 Da. Ferritin was not observed in Tt. foetus, nor in two other anaerobic protists, Entamoeba histolytica and Giardia intestinalis. Analysis of Tt. foetus hydrogenosomes showed at least nine iron-binding compounds, which were absent in metronidazole-resistant mutants. The major iron-binding compound was identified as [2Fe-2S] ferredoxin of the adrenodoxin type.