Methods in enzymology最新文献

While tRNA-derived fragments (tDRs) play important roles in gene expression regulation, it is technically challenging to distinguish bona fide tDRs from nicked tRNAs. This is because analytical techniques used to study RNA, such as northern blot, RT-qPCR or sequencing involve the use of denaturing reagents (e.g., phenol, formamide, urea) or physical procedures (e.g., heat) that convert nicked tRNAs into tRNA halves or other tDRs. In this chapter, we describe a protocol that enables the purification of nicked tRNAs under non-denaturing conditions that preserve their 3D structure. Purified nicked tRNAs can then be either enzymatically repaired into almost full-length tRNAs, or chromatographically separated from single-stranded tDRs before detection. These protocols will allow researchers to distinguish between structurally distinct but sequence identical tDRs and nicked tRNAs, disentangling their biological functions.

Cofactor imbalance is a common challenge in whole-cell bioconversion and thus limits the efficiency of biocatalysts. Various approaches have been employed to enhance cofactor availability, including specific engineering of pathways to increase intracellular levels of NAD(P)H, FMN, FAD, ATP and CoA. Recently, we have demonstrated that addition of xylose reductase (XR) in and supplying lactose to metabolically engineered cells can enhance levels of their sugar phosphates, leading to greater synthesis of NAD(P)H, FMN, FAD, ATP, and CoA in these cells, and thus a higher yield of bioconversion products. We propose that the XR/lactose system can be used as a generic tool to enhance precursor pools for cofactor synthesis for various in vivo biocatalysts. Here, we provide a protocol for the use of the XR/lactose system in fatty alcohol biosynthesis by Escherichia coli BL21(DE3). Step-by-step protocols and remarks should allow readers to adapt the use of XR/lactose for their engineered cells which should alleviate the problem of cofactor supply in whole-cell biocatalysis.

Climate change is an urgent and collective challenge, and new processes to synthesize complex molecules in a more sustainable way are highly desirable. Biocatalysis can be a strong player in this field, due to the specificity of enzymes and their ability to catalyze complex reactions at mild conditions. However, these reactions often require the regeneration of expensive cofactors in order to obtain relevant amounts of product. In vivo biocatalysis offers a solution to this problem by plugging the reaction in the microbial metabolism, which supplies the necessary energy. In particular, Cupriavidus necator H16 (C. necator H16) is an attractive microbial chassis due to its versatility and its lithoautotrophic metabolism. Its O₂-tolerant soluble hydrogenase (SH) can be used to regenerate nicotinamide cofactors in an atom-efficient manner, without the creation of undesired side products. This hydrogenase has already been used as a cofactor regeneration system in vitro, but examples of in vivo biocatalysis are scarce due to the time-consuming genetic engineering process of C. necator H16. In this book chapter, we present a strategy for the engineering of C. necator from plasmid cloning (using a recently developed expression plasmid) to protein expression of a model oxidoreductase. This pipeline allows for rapid and streamlined strain engineering, which can aid the discovery and development of future in vivo biocatalytic processes using C. necator H16.

RNA modifications are key regulators for RNA processes. tRNA-derived RNAs are small RNAs with size between 15 and 50 bases long that are processed from mature or precursor tRNAs. Despite their more recent discovery, tRNA-derived RNAs have been found to play regulatory roles in many cellular processes including gene silencing, protein synthesis, stress response, and transgenerational inheritance. Furthermore, tRNA-derived RNAs are highly abundant in bodily fluids, posing as potential biomarkers. A unique feature of tRNA-derived RNAs is that they are rich in RNA modifications. Many of the RNA modifications on tRNA-derived RNAs disrupt Watson-Crick base pairing and will thus stall reverse transcriptase, such as N¹-methyladenosine (m¹A), N¹-methylguanosine (m¹G) and N², N²-dimethylguanosine (m²₂G). These RNA modifications add another layer of regulation onto tRNA-derived RNAs' functions and are of interests for future research. However, these RNA modifications could also lead to lower detection of modification-containing RNAs in genome-wide small RNA sequencing analysis due to reverse transcriptase stall. To circumvent this bias, TGIRT (Thermostable Group II Intron Reverse Transcriptase) has been used to readthrough RNA modifications inserting mismatches. These mismatch signatures can then be used to precisely map the modification sites at base resolution. Here we describe the step-by-step experimental protocol to start with purified RNAs from cells or tissues and use TGIRT to make small RNA sequencing library for Illumina sequencing to profile the abundance of tRNA-derived RNAs and the associated RNA modifications.

The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is involved in diverse biological processes. The APOBEC deaminase family acts in various cellular processes mostly through inducing C-to-U mutation in single-stranded RNA (or DNA). However, comparing the activity of different RNA editing enzymes to one another is difficult due to the limited number of systems that can provide direct and efficient readout. In this report, a system in which RNA editing directly prompts a change in the subcellular localization of a modified eGFP structure is described in detail. This approach allows us to compare relative fluorescence intensity based on the RNA editing level. When observed through a fluorescence detection system, like a scanning confocal microscope, the cellular nucleus can be readily identified using a DNA-binding stain, such as DAPI or Hoechst, so that the accurate calculation of the ratio of nuclear to cytosolic eGFP intensity can be applied for an individual cell. This method provides a useful and flexible tool to examine and quantify RNA editing activity within cells, and it is not only limited to APOBEC proteins, but can also be applied more generally to other RNA editing enzymatic assays.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in duplex RNA, and through the delivery of guide RNAs, can be directed to edit specific adenosine sites. As ADARs are endogenously expressed in humans, their editing capacities hold therapeutic potential and allow us to target disease-relevant sequences in RNA through the rationale design of guide RNAs. However, current design principles are not suitable for difficult-to-edit target sites, posing challenges to unlocking the full therapeutic potential of this approach. This chapter discusses how we circumvent this barrier through an in vitro screening method, En Masse Evaluation of RNA Guides (EMERGe), which enables comprehensive screening of ADAR substrate libraries and facilitates the identification of editing-enabling guide strands for specific adenosines. From library generation and screening to next generation sequencing (NGS) data analysis to verification experiments, we describe how a sequence of interest can be identified through this high-throughput screening method. Furthermore, we discuss downstream applications of selected guide sequences, challenges in maximizing library coverage, and potential to couple the screen with machine learning or deep learning models.

Polyamines, including putrescine, spermidine, and spermine, are organic cations essential for cell growth, proliferation, and tissue regeneration. Their levels are tightly regulated by a set of enzymes controlling their biosynthesis, catabolism, and interconversion. Dysregulation of polyamine metabolism is associated with a group of rare genetic neurodevelopmental disorders collectively known as "polyaminopathies", including Snyder-Robinson Syndrome (SRS). SRS is an X-linked recessive disorder caused by mutations in the SMS gene, which encodes the spermine synthase enzyme. The lack of spermine synthase leads to aberrant polyamine levels and neurological impairments, as observed in patients and animal models. Currently, there are no available treatment options for SRS. Due to its monogenic nature, SRS is an excellent candidate for gene replacement therapy. The recent success of Zolgensma in treating children with Spinal Muscular Atrophy and the establishment of Platform Vector Gene Therapy (Pave-GT) initiative at the National Institute of Health (NIH) offer a framework to adapt-and-apply the same gene delivery system for multiple rare disease gene therapies. This chapter outlines strategies for delivering a functional copy of the SMS gene using an adeno-associated viral (AAV) vector, as well as methods to evaluate the molecular efficacy of this approach in an SRS mouse model. Our ultimate goal is to establish a versatile platform for genetic interventions targeting SRS and other polyaminopathies.

Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.

The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.