Linear Templates Research Articles

Gene editing in Plasmodium berghei made easy: Development of a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation

Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600−700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.

Effective Use of Linear DNA in Cell-Free Expression Systems.

Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.

Rolling-circle and strand-displacement mechanisms for non-enzymatic RNA replication at the time of the origin of life

It is likely that RNA replication began non-enzymatically, and that polymerases were later selected to speed up the process. We consider replication mechanisms in modern viruses and ask which of these is possible non-enzymatically, using mathematical models and experimental data found in the literature to estimate rates of RNA synthesis and replication. Replication via alternating plus and minus strands is found in some single-stranded RNA viruses. However, if this occurred non-enzymatically it would lead to double-stranded RNA that would not separate. With some form of environmental cycling, such as temperature, salinity, or pH cycling, double-stranded RNA can be melted to form single-stranded RNA, although re-annealing of existing strands would then occur much faster than synthesis of new strands. We show that re-annealing blocks this form of replication at a very low concentration of strands. Other kinds of viruses synthesize linear double strands from single strands and then make new single strands from double strands via strand-displacement. This does not require environmental cycling and is not blocked by re-annealing. However, under non-enzymatic conditions, if strand-displacement occurs from a linear template, we expect the incomplete new strand to be almost always displaced by the tail end of the old strand through toehold-mediated displacement. A third kind of replication in viruses and viroids is rolling-circle replication which occurs via strand-displacement on a circular template. Rolling-circle replication does not require environmental cycling and is not prevented by toehold-mediated displacement. Rolling-circle replication is therefore expected to occur non-enzymatically and is a likely starting point for the evolution of polymerase-catalysed replication.

High-Efficiency Protection of Linear DNA in Cell-Free Extracts from Escherichia coli and Vibrio natriegens.

The field of cell-free synthetic biology is an emerging branch of engineered biology that allows for rapid prototyping of biological designs and, in its own right, is becoming a venue for the in vitro operation of gene circuit-based sensors and biomanufacturing. To date, the related DNA encoded tools that operate in cell-free reactions have primarily relied on plasmid DNA inputs, as linear templates are highly susceptible to degradation by exonucleases present in cell-free extracts. This incompatibility has precluded significant throughput, time and cost benefits that could be gained with the use of linear DNA in the cell-free expression workflow. Here to tackle this limitation, we report that terminal incorporation of Ter binding sites for the DNA-binding protein Tus enables highly efficient protection of linear expression templates encoding mCherry and deGFP. In Escherichia coli extracts, our method compares favorably with the previously reported GamS-mediated protection scheme. Importantly, we extend the Tus-Ter system to Vibrio natriegens extracts, and demonstrate that this simple and easily implemented method can enable an unprecedented plasmid-level expression from linear templates in this emerging chassis organism.

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 µg of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

In silico Design of Linear DNA for Robust Cell-Free Gene Expression.

Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.

Bridging from the Sequence to Architecture: Graft Copolymers Engineering via Successive Latent Monomer and Grafting‐from Strategies†

Main observation and conclusionThe on‐demand building copolymer structures, from sequence to architecture, is crucial in understanding the relation between polymer structure and property, meanwhile motivating the innovation of polymer hierarchy. However, the challenge is conspicuous for complicated polymer structures from inherently intricate polymerization. In this work, copolymers with tailored grafting density and distributions were achieved using successive latent monomer and grafting‐from strategies. The hydroxyl group functionalized furan/maleimide adduct (FMOH) was selected as the latent monomer for RAFT polymerization of an array of copolymers with tailored localization of hydroxyl group along the main chain. The hydroxyl group further initiated the ring opening polymerization (ROP) of L‐lactide or ε‐caprolactone, resulting in a library of multicomponent copolymers via grafting‐from strategy. The initiating efficiency reached to ~100% with variable molecular weight (21300—58600 Da) and narrow distributions (ÐM < 1.25), indicating that such graft copolymers possessed controlled density and distribution of side chains as its linear template. The investigation on thermal properties of the well‐defined graft copolymers implied that the precise tailoring over copolymer structures at the molecule level could lead to tunable chemical/physical properties. This work bridged polymer from sequence to architecture, unveiled a new method in creating graft copolymers with programmable structures and provided the insight into the structure/property relationship.

Controllable Design and Preparation of Hollow Carbon-Based Nanotubes for Asymmetric Supercapacitors and Capacitive Deionization.

Carbon-based materials are important desirable materials in areas such as supercapacitors and capacitive deionization. However, traditional commercial materials are heterogeneous and prone to agglomeration in nanoscale and have structural limitation of electrochemical and desalination performance due to poor transport channels and low capacitance of prepared electrodes. Here, we introduce the facile strategy for controllable preparation of two types of hollow carbon-based nanotubes (HCTs) with amorphous mesoporous structures, which are synthesized by employing a MnO2 linear template method and calcination of polymer precursors. The porous N-doped HCT (NHCT) shows a specific capacitance of 412.6 F g-1 (1 A g-1), with 77.3% rate capability (20 A g-1). The fabricated asymmetric MnO2//NHCT supercapacitor displays the energy density of 55.8 Wh kg-1 at a power density of 803.9 W kg-1. Furthermore, two typical MnO2//HCT and MnO2//NHCT devices both show the selective desalination performance of sulfate, and the MnO2//NHCT device possesses a high deionization value of 11.37 mg g-1 (500 mg L-1 Na2SO4). These fabricated hollow carbon-based architectures with functional characteristics promise potential applications in energy and environmental related fields.

MRI brain templates of the male Yucatan minipig

The pig is growing in popularity as an experimental animal because its gyrencephalic brain is similar to humans. Currently, however, there is a lack of appropriate brain templates to support functional and structural neuroimaging pipelines. The primary contribution of this work is an average volume from an iterative, non-linear registration of 70 five- to seven-month-old male Yucatan minipigs. In addition, several aspects of this study are unique, including the comparison of linear and non-linear template generation, the characterization of a large and homogeneous cohort, an analysis of effective resolution after averaging, and the evaluation of potential in-template bias as well as a comparison with a template from another minipig species using a “left-out” validation set. We found that within our highly homogeneous cohort, non-linear registration produced better templates, but only marginally so. Although our T1-weighted data were resolution limited, we preserved effective resolution across the multi-subject average, produced templates that have high gray-white matter contrast and demonstrate superior registration accuracy compared to an alternative minipig template.

Transformation of Borrelia burgdorferi.

Transformation techniques used to genetically manipulate Borrelia burgdorferi, the agent of Lyme disease, play a critical role in generating mutants that facilitate analyses of the role of genes in the pathophysiology of this bacterium. A number of borrelial mutants have been successfully isolated and characterized since the first electrotransformation procedure was established 25 years ago (Samuels, 1995). This article is directed at additional considerations for transforming infectious B. burgdorferi to generate strains retaining the plasmid profile of the parental strain, enabling analysis of transformants for in vitro and in vivo phenotypes. These methods are built on previously published protocols and are intended to add steps and tips to enhance transformation efficiency and recovery of strains amenable for studies involving colonization, survival, and transmission of B. burgdorferi during the vector and vertebrate phases of infection. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of stock cultures, propagation of spirochetes, and analysis of plasmid profiles Basic Protocol 2: Preparation of plasmid and linear DNA templates for transformation Basic Protocol 3: Transformation of B. burgdorferi Basic Protocol 4: Antibiotic selection of borrelial transformants Basic Protocol 5: Isolation of borrelial transformants in agar overlays Basic Protocol 6: Complementation of mutant borrelial strains in cis or in trans.

A Tracking Registration Method for Augmented Reality Based on Multi-modal Template Matching and Point Clouds

In order to overcome the defects where the surface of the object lacks sufficient texture features and the algorithm cannot meet the real-time requirements of augmented reality, a markerless augmented reality tracking registration method based on multi-modal template matching and point clouds is proposed. The method first adapts the linear parallel multi-modal LineMod template matching method with scale invariance to identify the texture-less target and obtain the reference image as the key frame that is most similar to the current perspective. Then, we can obtain the initial pose of the camera and solve the problem of re-initialization because of tracking registration interruption. A point cloud-based method is used to calculate the precise pose of the camera in real time. In order to solve the problem that the traditional iterative closest point (ICP) algorithm cannot meet the real-time requirements of the system, Kd-tree (k-dimensional tree) is used under the graphics processing unit (GPU) to replace the part of finding the nearest points in the original ICP algorithm to improve the speed of tracking registration. At the same time, the random sample consensus (RANSAC) algorithm is used to remove the error point pairs to improve the accuracy of the algorithm. The results show that the proposed tracking registration method has good real-time performance and robustness.

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Guidelines for nucleic acid template design for optimal cell-free protein synthesis using an Escherichia coli reconstituted system or a lysate-based system.

Cell-free protein synthesis is an attractive method for generating enzyme/protein variants for simplified functional analysis as both in vitro protein expression and analysis may often be performed in a single vial or well. Today, researchers may choose from multiple commercial cell lysate products or reconstituted systems which are compatible with either mRNA, linear DNA or plasmid DNA templates. Here we provide guidance for optimal design of the genetic elements within linear and plasmid DNA templates which are required to reliably practice cell-free protein synthesis. Protocols are presented for generating linear DNA templates, and data are presented to show that linear DNA templates may in many cases provide robust protein yields even when employing an Escherichia coli lysate for protein synthesis. Finally, the use of linear DNA templates makes it possible to bypass all cell cultivation steps and proceed from PCR amplification of synthetic DNA to generation of target protein in a matter of hours.

Cell-free protein synthesis of CRISPR ribonucleoproteins (RNP).

Cell-free protein synthesis is a powerful tool to produce recombinant proteins, and as an open system, can often integrate all or part of downstream assays. Here we describe in vitro synthesis of the Streptococcus pyogenes type II-A CRISPR-Cas9 ribonucleoproteins (SpCas9 RNPs), consisting of the effector protein and the single guide RNAs (sgRNAs). In spite of its large molecular weight (160kDa), the SpCas9 effector is expressed relatively well from linear DNA templates under T7 promoter in commercial reconstituted cell-free protein synthesis systems. sgRNAs can be added before the effector synthesis reaction, or transcribed directly from linear DNA templates during the synthesis reaction. The newly synthesized SpCas9 effector forms an active RNP complex with sgRNAs. When a reconstituted cell-free system is used, the target DNA templates can also be added in the reactions, thereby combining CRISPR-Cas synthesis and activity assay.

Multicolor fluorescence encoding of different microRNAs in lung cancer tissues at the single-molecule level.

The simultaneous detection of multiple microRNAs (miRNAs) will facilitate early clinical diagnosis. Herein, we demonstrate the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection for simultaneous measurement of different miRNAs in lung cancer tissues. This assay involves two linear templates and two circular templates without the requirement of any fluorescent-labeled probes. The binding of target miRNAs to their corresponding linear templates initiates the cyclic strand displacement amplification, generating many triggers which can specifically hybridize with the corresponding biotin-labeled AP probes to initiate the apurinic/apyrimidic endonuclease 1-assisted cyclic cleavage reaction for the production of more biotin-labeled primers for each miRNA. The resultant two primers can react with their corresponding circular templates to initiate rolling circle amplification which enables the incorporation of Cy5-dCTP/Cy3-dGTP nucleotides, resulting in the simultaneous production of abundant biotin-/multiple Cy5/Cy3-labeled DNA products. After magnetic separation and exonuclease cleavage, the amplified products release abundant Cy5 and Cy3 fluorescent molecules which can be simply monitored by single-molecule detection, with Cy3 indicating miR-21 and Cy5 indicating miR-155. This assay involves three consecutive amplification reactions, enabling the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophore-encoded DNA products which can release abundant fluorescent molecules for the generation of amplified signals. This assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and in lung cancer tissues, and it can distinguish the expression of miRNAs between non-small cell lung cancer patients and healthy persons. The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction.

Highly improved cloning efficiency for plasmid-based CRISPR knock-in in C. elegans.

Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in C. elegans (Dickinson et al. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin et al. 2018; Ghanta and Mello 2020), the cloning required for plasmid-based protocols has been cited as a drawback of this knock-in approach. Using thorough quantitative assessment, we have found that cloning efficiency can reproducibly reach 90% for the plasmids of the self-excising cassette (SEC) selection method, essentially resolving cloning as a burden for plasmid-based CRISPR knock-in.

Bimodal Mesoporous Carbon Spheres with Small and Ultra-Large Pores Fabricated Using Amphiphilic Brush Block Copolymer Micelle Templates

We report the self-assembly of amphiphilic polystyrene-block-poly(ethylene oxide) (PS-b-PEO) brush block copolymers (BBCPs) into spherical micelles in an ethanol/water mixture as an efficient templating approach to fabricate mesoporous carbon spheres using polydopamine as a carbon source. Mesopore sizes of up to 25 nm are well controlled and are dependent on the molecular weight (Mw) of the BBCP. Such large pores are difficult to obtain using traditional linear block copolymers templates. Furthermore, bimodal mesoporous carbon spheres with two populations of pore sizes (24.5 and 6.5 nm) are obtained using a BBCP coassembled with a small molecule surfactant (Pluronic F127). An oxygen reduction reaction is used to demonstrate that electrocatalytic performance can be tuned by controlling the carbon sphere morphologies. This work provides a novel and versatile method to fabricate carbon spheres with broadly tunable bimodal pore sizes for potential applications in catalysis, separations, and energy storage.

CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.

The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.

Engineering Micrometer-Sized DNA Tracks for High-Speed DNA Synthesis and Biosensing.

φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote "micrometer-sized DNA track" (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer-binding domain at its 3' end and ≈1000 repeating sequence units at its 5' end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1 pM let-7a in 10 minutes.