Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore ...expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9–based genome editing as a potential therapy to treat DMD.
We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, ...reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed‐batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384‐well plates), 100 μl (96‐well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5–7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed‐batch fermentation.
Highlights
Stationary phase protein expression results in high titers.
Autoinduction by phosphate depletion enables protein titers from 2 to 8 g/L.
Autoinduction has been validated from 384‐well plates to instrumented bioreactors.
We report the scalable production of recombinant proteins in E. coli, reliant on autoinduction, triggered by phosphate depletion in stationary phase. In fed batch fermentations protein titers up to 8.1+/−0.7 g/L (˜270 mg/gCDW) are achieved. The process has been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μL (384 well plates), 100 μL (96 well plates), 20 mL and 100 mL, offering protein titers above 2 g/L at these smaller scales.
We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare ...two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.
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•Decreases in NADPH pools lead to increased NADPH fluxes.•Pyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo.•Dynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH flux.•Xylitol titers >200 g/L in fed batch fermentations with xylose as a sole feedstock.
We report that two-stage dynamic control improves bioprocess robustness as a result of the dynamic deregulation of central metabolism. Dynamic control is implemented during stationary phase using ...combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. Deregulated networks are less sensitive to environmental conditions improving process robustness. Process robustness in turn leads to predictable scalability, minimizing the need for traditional process optimization. We validate process robustness and scalability of strains and bioprocesses synthesizing the important industrial chemicals alanine, citramalate and xylitol. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology.
•Two-stage dynamic metabolic control enables deregulation of central metabolism.•Deregulation of the metabolic network improves process robustness.•Process robustness improves scalability from 96 well plates to instrumented bioreactors.•Rapid scale up from lab bioreactors to pilot bioreactors.
2-Stage microfermentations Li, Shuai; Ye, Zhixia; Moreb, Eirik A. ...
Metabolic engineering communications,
06/2024, Volume:
18, Issue:
C
Journal Article
Peer reviewed
Open access
Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It ...has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of protocols for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, by detailing the approach to protocol development, these methods are also adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.
•The development of high throughput, 2-stage microfermentations for microbial strain evaluation.•Protocols for both washing of cells between stages as well as automatic transitions.•The impact of key process considerations are discussed.
We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of ...both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two‐stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X‐100) and a single 30 min freeze‐thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high‐throughput cultivation in microtiter plates, and larger scale stirred‐tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.
Autolysis and autohydrolysis are enabled by induction of a periplasmic nuclease and cytoplasmic lysozyme, which after a membrane disruption lead to DNA/RNA hydrolysis and peptidoglycan degradation.
CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target ...site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.
CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction ...algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here, we perform a meta-analysis of 39 published CRISPR-Cas9 data sets to understand better the role of secondary structures in inhibiting gRNA activity. We (1) identify two distinct inhibitory structures that can form, (2) measure the prevalence of these structures in existing gRNA library data sets, and (3) provide free energy cutoffs at which these structures become inhibitory. First, we show that hairpins that form within the targeting portion (spacer) of the gRNA, having a minimum free energy of <-5 kcal/mol, negatively impact gRNA activity. Second, we demonstrate that a longer hairpin can form between the spacer and the nexus portion of the gRNA scaffold. A duplex stability of this longer hairpin of <-15 kcal/mol negatively impacts gRNA activity. These cutoffs help to explain conflicting impacts of free energy values in different data sets, as well as provide a guideline for future gRNA designs.
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•Comparison of biocatalysts/bioprocesses with traditional chemical catalysts/processes.•Turnover rates of biocatalysts can exceed those of chemical catalysts.•2-Stage bioprocesses ...offer the potential for greatly improved rates.•Dynamic metabolic control strategies can improve performance from 10% to over 1000%.•2-Stage dynamic metabolic control can lower bioprocess operating and capital costs.
Bioprocessing technology offers a potentially promising and more sustainable alternative to many traditional chemical process technologies; however to date this potential has largely not been realized. For large-scale bioprocessing to have larger penetration into larger volume chemical markets, new technologies need to both exploit the key advantages of biocatalysts over chemical catalysts, while addressing the key limitations of bioprocessing as compared to more traditional chemical process technology. The use of dynamic metabolic control strategies to engineer productive and robust stationary phase biocatalysts in combination with advanced two-stage fermentation has the potential to both increase process level metrics, including specific productivity, volumetric rates, titers and yields, while leveraging the unique ability of whole cell biocatalysts to perform multiple complex chemical conversions in a single unit operation.
CRISPR-based interference has become common in various applications from genetic circuits to dynamic metabolic control. In
, the native CRISPR Cascade system can be utilized for silencing by deletion ...of the
nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of spacer sequences from guide arrays utilized for dynamic silencing. We report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of spacers from guide arrays. Subsequently, mutant guide arrays can be amplified through selection. Replacing a constitutive promoter driving Cascade complex expression with a tightly controlled inducible promoter improves guide array stability, while minimizing leaky gene silencing. Additionally, these results demonstrate the potential of Cas1/2 mediated guide deletion as a mechanism to avoid CRISPR based autoimmunity.
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