Linear DNA Templates Research Articles

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template – encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage – expressed in the ‘protein synthesis using recombinant elements’ (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

In silico PCR analysis: a comprehensive bioinformatics tool for enhancing nucleic acid amplification assays.

Nucleic acid amplification assays represent a pivotal category of methodologies for targeted sequence detection within contemporary biological research, boasting diverse utility in diagnostics, identification, and DNA sequencing. The foundational principles of these assays have been extrapolated to various simple and intricate nucleic acid amplification technologies. Concurrently, a burgeoning trend toward computational or virtual methodologies is exemplified by in silico PCR analysis. In silico PCR analysis is a valuable and productive adjunctive approach for ensuring primer or probe specificity across a broad spectrum of PCR applications encompassing gene discovery through homology analysis, molecular diagnostics, DNA profiling, and repeat sequence identification. The prediction of primer and probe sensitivity and specificity necessitates thorough database searches, accounting for an optimal balance of mismatch tolerance, sequence similarity, and thermal stability. This software facilitates in silico PCR analyses of both linear and circular DNA templates, including bisulfited treatment DNA, enabling multiple primer or probe searches within databases of varying scales alongside advanced search functionalities. This tool is suitable for processing batch files and is essential for automation when working with large amounts of data.

Understanding the impact of in vitro transcription byproducts and contaminants.

The success of messenger (m)RNA-based vaccines against SARS-CoV-2 during the COVID-19 pandemic has led to rapid growth and innovation in the field of mRNA-based therapeutics. However, mRNA production, whether in small amounts for research or large-scale GMP-grade for biopharmaceutics, is still based on the In Vitro Transcription (IVT) reaction developed in the early 1980s. The IVT reaction exploits phage RNA polymerase to catalyze the formation of an engineered mRNA that depends on a linearized DNA template, nucleotide building blocks, as well as pH, temperature, and reaction time. But depending on the IVT conditions and subsequent purification steps, diverse byproducts such as dsRNA, abortive RNAs and RNA:DNA hybrids might form. Unwanted byproducts, if not removed, could be formulated together with the full-length mRNA and cause an immune response in cells by activating host pattern recognition receptors. In this review, we summarize the potential types of IVT byproducts, their known biological activity, and how they can impact the efficacy and safety of mRNA therapeutics. In addition, we briefly overview non-nucleotide-based contaminants such as RNases, endotoxin and metal ions that, when present in the IVT reaction, can also influence the activity of mRNA-based drugs. We further discuss current approaches aimed at adjusting the IVT reaction conditions or improving mRNA purification to achieve optimal performance for medical applications.

Supercoiled DNA percentage: A key in-process control of linear DNA template for mRNA drug substance manufacturing

The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high quality in-vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of polyA tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity, and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through Oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template CMC (Chemistry, Manufacturing, and Controls) and process development for mRNA drug substance.

Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3′ and 5′ ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3′ poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

An Idiotype Vaccine Strategy Utilizing Targeted NGS and Cell Free Protein Synthesis System Ensures Identification and Production of Human Idiotype Antigens, and Shows Promising Results in Preclinical Models of B-Cell Lymphoma with a Novel TLR7/8 Agonist-Based Adjuvant and Anti-PD-L1 Antibody

Background: Non-Hodgkin lymphoma (NHL) is one of the most common hematological malignancies in the world. Despite advances in treatment options, such as chimeric antigen receptor T cells (CAR-T) or bispecific antibodies, many patients still recur after complete remissions (CR) or do not achieve CR. Since B cell malignancies arise from clonal expansion, the B cell receptor (BCR) expressed on the cell surface, idiotype, has been studied as a tumor-specific antigen. Several trials have evaluated idiotype vaccines in clinical setting, but failed to demonstrate efficacy. Limitations have arisen from the uncertainty of BCR sequence confirmation, long turnaround time in manufacturing idiotype antigen, and the absence of strong immune modulators. Here, we utilized targeted next generation sequencing (NGS) for BCR, cell-free protein synthesis (CFPS) system for idiotype antigen manufacturing, and a novel liposomal TLR7/8 agonist-based adjuvant to optimize idiotype vaccine. Methods: To generate the idiotype antigens as single-chain variable fragment (scFv) from A20 lymphoma cell line and biopsy samples from NHL patients, variable regions of BCR heavy and light chain were sequenced by targeted NGS. Total RNA was isolated from A20 cells and tumor tissues from patients, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using constant region-specific primers, resulting in the 1 st strand cDNA. Subsequently, the 2 nd strand cDNA was synthesized using V-gene specific primers and sequenced using an Illumina MiSeq sequencer. We applied targeted NGS to patients' tumor tissues from Asan Medical Center and Seoul National University Hospital to understand whether this method works in human tissues as well. The identified sequence was cloned into a linear DNA template and expressed by CFPS system. We optimized the CFPS system and purification process of idiotype antigen scFv from A20 cell line and human tissues. The liposomal adjuvant ProLNG-001 (TLR7/8 agonist 140 μg) and ProLNG-002 (TLR7/8 agonist 70 μg + TLR4 agonist 5 ug) were synthesized by microfluidics method. Idiotype antigen A20-scFv 50 μg was given alone or mixed with ProLNG-001 or -002. To investigate the efficacy of the idiotype vaccine, BALB/c mice were subcutaneously injected with 5×10 5 of A20 cells. Mice were subcutaneously vaccinated 4 times at 1-week intervals starting from day 2 after tumor inoculation. Atezolizumab 10 mg/kg was administered intraperitoneally 3 times a week for 3 weeks in the idiotype vaccine + immune checkpoint inhibitor combination group (Figure 1a). Results: Targeted NGS has confirmed BCR sequences from A20 cell line and dominant malignant B cell clone from patients' tumor with a success rate of over 90% when sufficient volume (more than 500 mm 3) of biopsy sample was provided for RNA extraction. Also, we were able to express the idiotype scFv antigens determined from 18 NHL patients' tumor samples using CFPS system with a 100% success rate (Figure 1b). The untreated mice (Group 1) showed a tumor growth pattern similar to that of Group 2 (vaccination with antigen only). Vaccination with idiotype antigen mixed with liposomal adjuvants, ProLNG-001 (Group 3) and ProLNG-002 (Group 4), significantly inhibited tumor growth. The combination of anti-PD-L1 antibody and idiotype antigen mixed with ProLNG-002 (Group 5) generated even better overall anti-tumor responses with 14 out of 15 mice showing no residual tumor at day 34 after tumor inoculation. Conclusion: Taken together, the idiotype vaccine strategy using targeted NGS for BCR, CFPS system for idiotype antigen manufacturing, and TLR7/8-agonist-based liposomal adjuvants showed highly effective results in the preclinical syngeneic A20 tumor model. Furthermore, the usage of immune checkpoint inhibitors combined with idiotype vaccine in B cell lymphoma may exert synergistic effects. The CFPS system can be applied to swift, robust and personalized manufacturing of idiotype antigen for individuals with different idiotype sequence in a GMP setting, which was not possible in prior studies. These results support further development of the idiotypic vaccine as a treatment option for non-Hodgkin's B cell lymphomas.

Screening of Aptamers that Bind to the Multivalent Aminoglycoside Amikacin

Increased awareness of the multiple roles of RNA molecules has led to the realization that, in addition to their structural and functional roles, RNAs can be drug targets for small molecular therapy. Amikacin, a member of the aminoglycoside group of antibiotics, binds to specific sites in bacterial 16S ribosomal RNAs (rRNAs) and interferes with protein synthesis leading to cell death. Here, we used the systemic evolution of ligands by exponential enrichment (SELEX) method to isolate high affinity RNA fragments (aptamers) that bind to amikacin. After five rounds of SELEX selection, in which a linear N25 DNA template was used for the first selection cycle, the resulting RNA was cloned and sequenced. Among the 38 clones generated, five groups of sequences (groups A through E) containing nine conserved motifs were identified. The sequences of groups A and B were almost identical, indicating that the selected RNA was enriched. Subsequently, the Basic Local Alignment Search Tool program was used to search for the conserved motifs in bacterial 16S rRNA sequences. Strikingly, no sequence homology was observed, suggesting that the conserved sequences (motifs) identified in this studymay be novel target sites for amikacin. Bangladesh Pharmaceutical Journal 26(2): 134-143, 2023 (July)

T7Max transcription system

BackgroundEfficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.ResultsHere we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.ConclusionsThe modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Enhanced production of d-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR–Cpf1 genome editing method

BackgroundCorynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum.ResultsIn this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only.ConclusionsWe developed a RecET-assisted CRISPR–Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles.

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNpencapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNpis produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNpis subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNpnanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Akaby-Cell-free protein expression system for linear templates.

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

Constructing Cell-Free Expression Systems for Low-Cost Access.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203–424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

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.

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.

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.