Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Precise DNA cleavage using CRISPR-SpRYgests.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

A new family of CRISPR-type V nucleases with C-rich PAM recognition.

Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.

RNase H-based analysis of synthetic mRNA 5' cap incorporation.

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

Characterization of Cme and Yme thermostable Cas12a orthologs.

CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.

Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells.

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation.

Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles.

The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Francisella novicida Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium Bacillus smithii as our experimental platform, we demonstrate that FnCas12a is active in vivo at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for B. smithii, combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of B. smithii cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9.

Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.

Staphylococcus aureus Cas9 is a multiple-turnover enzyme.

Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics, we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.

mRNA capping: biological functions and applications.

The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.

Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

High-throughput sequencing (HTS) has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA) and other small RNAs (sRNA). Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

Active stabilization of human endothelial nitric oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs.

Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3' untranslated region (3' UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3'-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3'-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3'-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.

Small RNA expression profiling by high-throughput sequencing: implications of enzymatic manipulation.

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.

Functional importance of Dicer protein in the adaptive cellular response to hypoxia.

The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.

Structural bias in T4 RNA ligase-mediated 3'-adapter ligation.

T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3'-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3'-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3'-adapter ligation step.

Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA.

Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2'-O-methylation of the 3'-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.

Endothelial nitric oxide synthase gene expression during murine embryogenesis: commencement of expression in the embryo occurs with the establishment of a unidirectional circulatory system.

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with beta-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.

RNA helicase A interacts with RISC in human cells and functions in RISC loading.

RNA interference is a conserved pathway of sequence-specific gene silencing that depends on small guide RNAs and the action of proteins assembled in the RNA-induced silencing complex (RISC). Minimally, the action of RISC requires the endonucleolytic slicer activity of Argonaute2 (Ago2) directed to RNA targets whose sequences are complementary to RISC-incorporated small RNA. To identify RISC components in human cells, we developed an affinity-purification strategy to isolate siRNA-programmed RISC. Here we report the identification of RNA helicase A (RHA) as a human RISC-associated factor. We show that RHA interacts in human cells with siRNA, Ago2, TRBP, and Dicer and functions in the RNAi pathway. In RHA-depleted cells, RNAi was reduced as a consequence of decreased intracellular concentration of active RISC assembled with the guide-strand RNA and Ago2. Our results identify RHA as a RISC component and demonstrate that RHA functions in RISC as an siRNA-loading factor.