Highlights ► Select immune markers may be correlates of immunity after vaccination. ► Polyfunctional T cells are indicators of a high quality immune response. ► Peptide loaded MHC tetramers identify ...antigen-specific cytotoxic T cells. ► Humoral immune response markers remain the only validated correlates of protection.
CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome ...engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.
•Multiplex CRISPR/Cas9 procedure for 1–5 marker-free genome edits in S. cerevisiae.•Genome re-sequencing shows that the tool is likely to have no off-target effects.•Generation of all strain combinations using 5 targets without selected phenotype.•Identification of strains with increased mevalonate levels of >41-fold compared to reference.
In this paper, we review the emerging concept of digital twins (DTs) for urban water systems (UWS) based on the literature, stakeholder interviews and analyzing the current DT implementation process ...in the utility company VCS Denmark (VCS). Here, DTs for UWS are placed in the context of DTs at the component, unit process/operation or hydraulic structure, treatment plant, system, city, and societal levels. A UWS DT is characterized as a systematic virtual representation of the elements and dynamics of the physical system, organized in a star-structure with a set of features connected by data links that are based on standards for open data. This allows the overall functionality to be broken down into smaller, tangible units (features), enabling microservices that communicate via data links to emerge (the most central feature), facilitated by application programing interfaces (APIs). Coupled to the physical system, simulation models and advanced analytics are among the most important features. We propose distinguishing between living and prototyping DTs, where the term “living” refers to coupling observations from an ever-changing physical twin (which may change with, e.g., urban growth) with a simulation model, through a data link connecting the two. A living DT is thus a near real-time representation of an UWS and can be used for operational and control purposes. A prototyping DT represents a scenario for the system without direct coupling to real-time observations, which can be used for design or planning. By acknowledging that different DTs exist, it is possible to identify the value-creation from DTs achieved by different end-users inside and outside a utility organization. Analyzing the DT workflow in VCS shows that a DT must be multifunctional, updateable, and adjustable to support potential value creation across the utility company. This study helps clarify key DT terminology for UWS and identifies steps to create a DT by building upon digital ecosystems (DEs) and open standards for data.
Here we describe a method for robust directed evolution using mutagenesis of large sequence spaces in their genomic contexts. The method employs error-prone PCR and Cas9-mediated genome integration ...of mutant libraries of large-sized donor variants into single or multiple genomic sites with efficiencies reaching 98–99%. From sequencing of genome integrants, we determined that the mutation frequency along the donor fragments is maintained evenly and successfully integrated into the genomic target loci, indicating that there is no bias of mutational load towards the proximity of the double strand break. To validate the applicability of the method for directed evolution of metabolic gene products we engineered two essential enzymes in the mevalonate pathway of Saccharomyces cerevisiae with selected variants supporting up to 11-fold higher production of isoprenoids. Taken together, our method extends on existing CRISPR technologies by facilitating efficient mutagenesis of hundreds of nucleotides in cognate genomic contexts.
•Method for CRISPR/Cas9-mediated directed evolution of large sequences in genomic contexts.•Method works with > 98% efficiency to target directed evolution of essential genes.•Method supports integration of large donor variants without biases towards double strand breaks.•Method supports genome integration of thousands of donor variant fragments in a multiplex manner.•Method was validated by high-throughput optimization of isoprenoid production.
Background and purpose - Using contemporary indications, up to 50% of patients undergoing knee arthroplasty are eligible for unicompartmental knee arthroplasty (UKA), and lower UKA use likely ...reflects a restrictive approach to patient selection. Since broader indications have been successfully introduced, and low surgical volume and UKA percentage (usage) are associated with higher revision rates, it is of interest whether the actual use of UKA has changed accordingly. We explored this by assessing time trends in patient demographics and whether these are associated with center UKA volume and usage.
Patients and methods - From the Danish Knee Arthroplasty Registry, we included 8,501 medial UKAs performed for primary osteoarthritis during 2002-2016. Using locally weighted regression, we examined changes-both overall and by center volume and usage (low vs high)-in sex distribution, age, weight, and preoperative American Knee Society Score (AKSS-O).
Results - Over the last 20 years, UKA use in Denmark has been increasing steadily. Age, weight, and proportion of men all increased regardless of volume and usage. AKSS-O showed an initial increase followed by a decrease. In low-usage and low-volume centers, the proportion of women was higher, patients were younger, weighed less, and had higher AKSS-O scores; however, for age and AKSS-O, the groups were converging during the last part of the period.
Interpretation - Characteristics of UKA patients have changed in the last 15 years irrespective of center volume and usage. We found between-group differences for both volume and usage, though with convergence for age and AKSS-O, which suggests an increasingly uniform approach to patient selection.
Simultaneous targeting of different antigens by bispecific antibodies (bsAbs) is permitting synergistic binding functionalities with high therapeutic potential, but is also rendering their analysis ...challenging. We introduce flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs with diverse molecular architectures and valencies under near-native conditions without potentially obstructive surface immobilization. Individual equilibrium dissociation constants are determined in solution, even in higher-order complexes with both antigens involved, hereby allowing the analysis of binding cooperativity and elucidation of a potential interference between the interactions. We further illustrate bispecific binding functionality as incremental increases in complex sizes when the bsAbs are exposed to one or two antigens. The possibility for comprehensive binding analysis with low material consumption and high matrix tolerability irrespective of molecular format and with little optimization renders FIDA a versatile tool for format selection and characterization of complex bi/multispecific protein therapeutics throughout the drug development and biomanufacturing pipeline.
The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable ...loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.
Large, randomized libraries are a key technology for many biotechnological applications. While genetic diversity is the main parameter most libraries direct their resources on, less focus is devoted ...to ensuring functional IN-frame expression. This study describes a faster and more efficient system based on a split β-lactamase complementation for removal of OFF-frame clones and increase of functional diversity, suitable for construction of randomized libraries. The gene of interest is inserted between two fragments of the β-lactamase gene, conferring resistance to β-lactam drugs only upon expression of an inserted IN-frame gene without stop codons or frameshifts. The preinduction-free system was capable of eliminating OFF-frame clones in starting mixtures of as little as 1% IN-frame clones and enriching to about 70% IN-frame clones, even when their starting rate was as low as 0.001%. The curation system was verified by constructing a single-domain antibody phage display library using trinucleotide phosphoramidites for randomizing a complementary determining region, while eliminating OFF-frame clones and maximizing functional diversity.
•Generation of libraries with randomized genes for various applications.•Goal is large, diverse libraries, but can result in many OFF-frame genes.•Improved, faster, robust version of split β-lactamase IN-frame enrichment system.•High enrichment rate, efficient, greater yield of IN-frame clones in shorter time.•Use of Nanopore and Sanger sequencing to prove enrichment efficiency.
The production of high‐value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing ...processes. The discovery of CRISPR‐Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next‐generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.
Bispecific antibodies (bsAbs) have attracted significant attention due to their dual binding activity, which permits simultaneous targeting of antigens and synergistic binding effects beyond what can ...be obtained even with combinations of conventional monospecific antibodies. Despite the tremendous therapeutic potential, the design and construction of bsAbs are often hampered by practical issues arising from the increased structural complexity as compared to conventional monospecific antibodies. The issues are diverse in nature, spanning from decreased biophysical stability from fusion of exogenous antigen-binding domains to antibody chain mispairing leading to formation of antibody-related impurities that are very difficult to remove. The added complexity requires judicious design considerations as well as extensive molecular engineering to ensure formation of high quality bsAbs with the intended mode of action and favorable drug-like qualities. In this review, we highlight and summarize some of the key considerations in design of bsAbs as well as state-of-the-art engineering principles that can be applied in efficient construction of bsAbs with diverse molecular formats.
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