Genotype-phenotype Linkage Research Articles

An enhanced Eco1 retron editor enables precision genome engineering in human cells from a single-copy integrated lentivirus.

Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast; however, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity as major contributors to poor retron editor efficiency. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to liberate the sgRNA and ncRNA. These modifications yielded a ncRNA with 5'- and 3'-end protection and an sgRNA with minimal 5' extension. This strategy increased steady-state ncRNA levels and rescued Cas9 activity leading to enhanced efficiency of the Eco1 retron editor in human cells. The enhanced Eco1 retron editor enabled the insertion of missense mutations in human cells from a single integrated lentivirus, thereby ensuring genotype-phenotype linkage over multiple cell divisions. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells. Here we present an enhanced Eco1 retron editor that enables efficient introduction of missense mutations in human cells from a single heritable genome copy.

PHEIGES: all-cell-free phage synthesis and selection from engineered genomes

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

YEAST SURFACE DISPLAY OF FULL-LENGTH IGG FOR ENGINEERING MONOCLONAL AND BISPECIFIC ANTIBODIES

Abstract The single-chain variable fragment (scFv) and antigen-binding fragment (Fab) are widely used in yeast surface display for antibody engineering. However, these non-IgG antibody fragments are not always well-expressed or functional. We developed a novel approach to display full-length IgG on yeast surfaces for engineering monoclonal and bispecific antibodies. The yeast surface display of full-length IgG was generated by transforming glycoengineered yeast P. pastoris with plasmids encoding for anchor protein, heavy chains, and light chains. IgG-displaying yeast cells were incubated with the fluorophore antigen and secondary detection antibody for FACS sorting. The displayed antibody with bound antigen and detection antibody was detected by FACS sorting based on both antibody affinity and display level. FACS sorting could eliminate the deviation caused by expression levels. The yeast surface display of IgG-like bispecific antibodies was also constructed, which exhibited specific binding of two distinct fluorophore antigens in flow-cytometry analysis. Thus, our yeast surface display of full-length IgG can be used to screen light chain libraries to isolate common light chains for bispecific antibodies. To assess antigen-binding efficiency between different displaying formats, an antibody was respectively displayed as full-length IgG or Fab on the surface of yeast cells for flow-cytometry analysis. Cells displaying full-length IgG showed a higher level of fluorescence than those displaying Fab fragments, indicating that yeast surface display of full-length IgG is superior for high-throughput screening. To test the specificity of the displayed antibody, we performed a demo experiment in which TNF-binding adalimumab-displaying cells were mixed with trastuzumab-displaying cells at a 1 to 1,000,000 ratio, mimicking the immune library. After three consecutive rounds of sorting, the yeast cells with high fluorescence were plated on a selective medium and the individual antibody clones were sequenced. All ten sequenced clones were confirmed to be adalimumab, demonstrating the maintenance of genotype-phenotype linkage for library screening in yeast surface display of full-length IgG. A mutation library was also generated from the initial hit and displayed at the surface of yeast cells for the screening of higher affinity maturation antibodies by FACS. A pool of high-affinity binders was plated on a selective medium. Individual clones were analyzed by flow cytometry and sequenced to identify unique antibodies with higher affinity. In summary, full-length IgG antibodies can be displayed on the yeast cell surface, mimicking their native forms in molecular structure and biophysical properties. The technique combines the high throughput of yeast display with mammalian-cell quality control. This novel approach can be used to engineer monoclonal and bispecific antibodies for high affinity and improved developability profiles.

Ribozyme activity modulates the physical properties of RNA-peptide coacervates.

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

Transcription-Associated Fluorescence-Activated Droplet Sorting for Di-rhamnolipid Hyperproducers.

Rhamnolipids (RLs) are biosurfactants with great economic significance that have been used extensively in multiple industries. Pseudomonas aeruginosa is a promising microorganism for sustainable RL production. However, current CTAB-MB based screening of RL-producing strains is time-consuming, labor-intensive, and unable to distinguish mono- and di-RL. In this study, we developed a novel transcription-associated fluorescence-activated droplet sorting (FADS) method to specifically target the di-RL hyperproducers. We first investigated critical factors associated with this method, including the specificity and sensitivity for discriminating di-RL overproducers from other communities. Validation of genotype-phenotype linkage between the GFP intensity, rhlC transcription, and di-RL production showed that rhlC transcription is closely correlated with di-RL production, and the GFP intensity is responsive to rhlC transcription, respectively. Using this platform, we screened out ten higher di-RL producing microorganisms, which produced 54-208% more di-RL than the model P.aeruginosa PAO1. In summary, the droplet-based microfluidic platform not only facilitates a more specific, reliable, and rapid screening of P.aeruginosa colonies with desired phenotypes, but also shows that intracellular transcription-associated GFP intensity can be used to measure the yield of di-RL between populations of droplets containing different environmental colonies. This method also can be integrated with transposon mutation libraries to target P.aeruginosa mutants.

Combinatorial InVitroFlow-assisted mutagenesis (CombIMut) yields a 41-fold improved CelA2 cellulase.

The combination of diversity generation methods and ultrahigh-throughput screening (uHTS) technologies is key to efficiently explore nature's sequence space and elucidate structure-function relationships of enzymes. Beneficial substitutions often cluster in a few regions and simultaneous amino acid substitutions at multiple positions (e.g., by OmniChange) will likely lead to further improved enzyme variants. An extensive screening effort is required to identify such variants, as the simultaneous randomization of four codons can easily yield over 105 potential enzyme variants. The combination of flow cytometer-based uHTS with cell-free compartmentalization technology using (w/o/w) double emulsions (InVitroFlow), provides analysis capabilities of up to 107 events per hour, thus enabling efficient screening. InVitroFlow is an elegant solution since diversity loss through a transformation of host cells is omitted and emulsion compartments provide a genotype-phenotype linkage through a fluorescence readout. In this study, a multisite saturation mutagenesisand an OmniChange library with four simultaneously saturated positions in the active site of CelA2 cellulase were screened using InVitroFlow. Screening of over 36 million events, yielded a significantly improved cellulase variant CelA2-M3 (H288F/H524Q) with an 8-fold increase in specific activity compared to the parent CelA2-H288F (83.9 U/mg) and a 41-fold increased specific activity (674.5 U/mg) compared to wildtype CelA2 (16.6 U/mg) for the substrate 4-MUC (4-methylumbelliferyl-β d-cellobioside).

Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Frontiers in single cell analysis: multimodal technologies and their clinical perspectives.

Single cell multimodal analysis is at the frontier of single cell research: it defines the roles and functions of distinct cell types through simultaneous analysis to provide unprecedented insight into cellular processes. Current single cell approaches are rapidly moving toward multimodal characterizations. It replaces one-dimensional single cell analysis, for example by allowing for simultaneous measurement of transcription and post-transcriptional regulation, epigenetic modifications and/or surface protein expression. By providing deeper insights into single cell processes, multimodal single cell analyses paves the way to new understandings in various cellular processes such as cell fate decisions, physiological heterogeneity or genotype-phenotype linkages. At the forefront of this, microfluidics is key for high-throughput single cell analysis. Here, we present an overview of the recent multimodal microfluidic platforms having a potential in biomedical research, with a specific focus on their potential clinical applications.

ABO B gene is associated with introversion personality tendancies through linkage with dopamine beta hydroxylase gene

Although personality is thought to be 50% heritable, consensus has not been reached about the specific genes involved, interest in genes affecting personality and behavior continues because of the linkage of personality traits with both physical and mental illness. One hundred and twenty years of study of the ABO blood types and the genes causing them has led to more precise assignment of genotype-phenotype linkage. Countries like Japan and Korea with 100years history of study of ABO blood groups and personality as well as other countries have published research with no consensus. Introversion is a well-studied personality trait with 50% genetic causation. Dopamine system has been linked to the introversion-extroversion spectrum with high dopamine linked to introversion. Dopamine beta hydroxylase determines the ratio of dopamine to norepinephrine. Dopamine beta hydroxylase gene, DBH, is in linkage disequilibrium with ABO gene thus offering insight into the ABO findings related to personality traits. ABO blood groups have been studied with some but not all studies finding type AB associated with introversion. One explanation for the inconsistent replication of findings could be that genotypes would show more differences than phenotypes. ABO A allele, ABO B allele or the synergism of both alleles could be the driver of introversion trait in ABO AB. Patients in an obstetric gynecology practice appeared to demonstrate a tendancy to introversion in phenotype ABO B. Since we had access to ABO blood phenotypes instead of genotypes of our obstetric gynecology office patients, we hypothesized that phenotype ABO AB when compared with ABO O would be associated with higher introversion since these phenotypes were also genotypes and since ABO O unlike ABO AB is not found associated with introversion in extant research. Though ABO B allele was observed to be the likely cause of introversion in patients and since we had available only phenotypes of ABO to use in search of genetic allele that causes introversion trait, ABO AB needs to be dissected to see whether ABO A allele or ABO B allele is the driver of the introspective trait. Based on the literature and on observation, the hypothesis was that ABO B is the driver of introversion trait. A pilot student of 225 obstetric gynecology patients using the online NPA personality test showed higher scores for introversion tendancies in ABO AB compared to ABO O and when ABO AB was compared to ABO A. This supports ABO B as the driver of introversion tendancies in the ABO gene. Studies using ABO genotypes instead of ABO phenotypes should provide further support for this hypothesis. Given the support for introversion tendancies in personality caused by higher dopamine genetically with dopamine beta hydroxylase low activity and this dopamine state being common mechanism for such conditions as schizophrenia and autism, continued discoveries of genes that impact this state will lead to many health implications.

The Multifaceted Effects of Serotonin Transporter Polymorphism (5-HTTLPR) on Anxiety, Implicit Moral Attitudes, and Harmful Behaviors.

Morality is fundamentally human in nature. Regardless, and even when moral norms seem to work toward the common goal of human cooperation, which morally contentious behaviors are permitted and which are prohibited vary across populations. Because of this occurrence, much scientific debate has revolved around the notion that this phenomenon might be explained by the interaction between genes and environment. Alongside, whether the principles cementing the bases of morality are intuition- or reason-based is another question that has been raised. However, previous research addressing these topics used explicit measures to probe moral attitudes, thus being the participants able to intentionally modify or disguise their honest responses. What’s more, while the 5-HTT gene was found to be associated with anxiety, morality, and even cultural structures, a single genotype–phenotype linkage cannot be established without considering the multifaceted effects of the 5-HTT gene on gene–behavior interactions. In order to explore the role of genetics on modeling moral attitudes and behaviors, we genotyped the 5-HTTLPR in 114 healthy volunteers and subsequently assessed their explicit justice sensitivity (Justice Sensitivity Inventory) and moral permissibility judgments, as well as their implicit moral attitudes [moral implicit association task (mIAT)]. Results revealed that 5-HTTLPR short-allele carriers had significantly lower mIAT reaction times when answering correctly and were less compliant on harming another person even when harm or death would inevitably occur anyway to this other individual. With these preliminary results, we can first see how it does not have to be a matter of vouching for a rationalist versus an intuitionist model of moral judgment, but rather being moral judgment an outcome of the different variants of the 5-HTTLPR polymorphism affecting the way in which individuals engage contrastingly with moral issues.

Generating Biologically Stable TNA Aptamers that Function with High Affinity and Thermal Stability.

Aptamers are often prone to nuclease digestion, which limits their utility in many biomedical applications. Here we describe a xeno-nucleic acid system based on α-l-threofuranosyl nucleic acid (TNA) that is completely refractory to nuclease digestion. The use of an engineered TNA polymerase permitted the isolation of functional TNA aptamers that bind to HIV reverse transcriptase (HIV RT) with KD's of ∼0.4-4.0 nM. The aptamers were identified using a display strategy that provides a powerful genotype-phenotype linkage. The TNA aptamers remain active in the presence of nuclease and exhibit markedly higher thermal stability than monoclonal antibodies. The combined properties of biological stability, high binding affinity, and thermal stability make TNA aptamers a powerful system for the development of diagnostic and therapeutic agents.

Systematic comparative analysis of single-nucleotide variant detection methods from single-cell RNA sequencing data

BackgroundSystematic interrogation of single-nucleotide variants (SNVs) is one of the most promising approaches to delineate the cellular heterogeneity and phylogenetic relationships at the single-cell level. While SNV detection from abundant single-cell RNA sequencing (scRNA-seq) data is applicable and cost-effective in identifying expressed variants, inferring sub-clones, and deciphering genotype-phenotype linkages, there is a lack of computational methods specifically developed for SNV calling in scRNA-seq. Although variant callers for bulk RNA-seq have been sporadically used in scRNA-seq, the performances of different tools have not been assessed.ResultsHere, we perform a systematic comparison of seven tools including SAMtools, the GATK pipeline, CTAT, FreeBayes, MuTect2, Strelka2, and VarScan2, using both simulation and scRNA-seq datasets, and identify multiple elements influencing their performance. While the specificities are generally high, with sensitivities exceeding 90% for most tools when calling homozygous SNVs in high-confident coding regions with sufficient read depths, such sensitivities dramatically decrease when calling SNVs with low read depths, low variant allele frequencies, or in specific genomic contexts. SAMtools shows the highest sensitivity in most cases especially with low supporting reads, despite the relatively low specificity in introns or high-identity regions. Strelka2 shows consistently good performance when sufficient supporting reads are provided, while FreeBayes shows good performance in the cases of high variant allele frequencies.ConclusionsWe recommend SAMtools, Strelka2, FreeBayes, or CTAT, depending on the specific conditions of usage. Our study provides the first benchmarking to evaluate the performances of different SNV detection tools for scRNA-seq data.

CDNA Display of Disulfide-Containing Peptide Library and In Vitro Evolution.

Directed in vitro evolution (IVE) is now a widely applied technology to obtain molecules that have designed new features of one's demands. We describe here experimental procedures for a cDNA display IVE to select peptide aptamers from a scaffold-based random peptide library. A three-finger (3-F) peptide library is exemplified, which has been shown its pluripotency to various target molecules. Peptide scaffolds including 3-F are refined through evolution, and they are mostly stabilized by disulfide bridges. To utilize such disulfide-containing protein library in IVE, we optimized the translation and folding conditions. Co-translational folding assisted by protein disulfide isomerase was found to have better efficiency than posttranslational refolding in the IVE. Linker is also a key element to make a tight genotype-phenotype linkage. Here, we introduced a whole procedure of IVE to use a newly designed puromycin linker, which was synthesized by a novel branching strategy. The improved linker enabled rapid and highly efficient ligation of mRNA and synthesis of protein fusions.

Ligand Engineering via Yeast Surface Display and Adherent Cell Panning.

High-throughput ligand discovery and evolution-via genotype-phenotype linkage strategies-empower molecularly targeted therapy, diagnostics, and fundamental science. Maintaining high-quality target antigen in these selections, particularly for membrane targets, is often a technical challenge. Panning yeast-displayed ligand libraries on intact mammalian cells expressing the molecular target has emerged as an effective strategy. Herein we describe the techniques used to select target-binding ligands via this approach including the use of target-negative cells to deplete non-specific binders and avidity reduction to preferentially select high-affinity ligands.

Selection strategies for randomly partitioned genetic replicators.

The amplification cycle of many replicators (natural or artificial) involves the usage of a host compartment, inside of which the replicator expresses phenotypic compounds necessary to carry out its genetic replication. For example, viruses infect cells, where they express their own proteins and replicate. In this process, the host cell boundary limits the diffusion of the viral protein products, thereby ensuring that phenotypic compounds, such as proteins, promote the replication of the genes that encoded them. This role of maintaining spatial colocalization, also called genotype-phenotype linkage, is a critical function of compartments in natural selection. In most cases, however, individual replicating elements do not distribute systematically among the hosts, but are randomly partitioned. Depending on the replicator-to-host ratio, more than one variant may thus occupy some compartments, blurring the genotype-phenotype linkage and affecting the effectiveness of natural selection. We derive selection equations for a variety of such random multiple occupancy situations, in particular considering the effect of replicator population polymorphism and internal replication dynamics. We conclude that the deleterious effect of random multiple occupancy on selection is relatively benign, and may even completely vanish is some specific cases. In addition, given that higher mean occupancy allows larger populations to be channeled through the selection process, and thus provide a better exploration of phenotypic diversity, we show that it may represent a valid strategy in both natural and technological cases.

Peptide barcoding for establishment of new types of genotype-phenotype linkages.

Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype–phenotype linkages.

Genetically Encoded Stimuli-Responsive Cytoprotective Hydrogel Capsules for Single Cells Provide Novel Genotype–Phenotype Linkage

Modification of cell surfaces with synthetic polymers is a promising approach for regulating cellular behavior. Here, we describe a genetically controlled strategy for selectively encapsulating sin...

Using single nucleotide variations in single-cell RNA-seq to identify subpopulations and genotype-phenotype linkage

Despite its popularity, characterization of subpopulations with transcript abundance is subject to a significant amount of noise. We propose to use effective and expressed nucleotide variations (eeSNVs) from scRNA-seq as alternative features for tumor subpopulation identification. We develop a linear modeling framework, SSrGE, to link eeSNVs associated with gene expression. In all the datasets tested, eeSNVs achieve better accuracies than gene expression for identifying subpopulations. Previously validated cancer-relevant genes are also highly ranked, confirming the significance of the method. Moreover, SSrGE is capable of analyzing coupled DNA-seq and RNA-seq data from the same single cells, demonstrating its value in integrating multi-omics single cell techniques. In summary, SNV features from scRNA-seq data have merits for both subpopulation identification and linkage of genotype-phenotype relationship.

Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics.

The success of ultrahigh-throughput screening experiments in directed evolution or functional metagenomics strongly depends on the availability of efficient technologies for the quantitative testing of a large number of variants. With advanced robotics, libraries of up to 105 clones can be screened per day as colonies on agar plates or cell lysates in microwell plates, albeit at high cost of capital, manpower and consumables. These cost considerations and the general need for high-throughput make miniaturization of assay volumes attractive. To provide a general solution to maintain genotype-phenotype linkage, biochemical assays have been compartmentalized into water-in-oil droplets. This chapter presents a microfluidic workflow that translates a frequently used screening procedure consisting of cytoplasmic/periplasmic protein expression and cell lysis to the single cell level in water-in-oil droplet compartments. These droplets are sorted based on reaction progress by fluorescence measurements at the picoliter scale.

Abnormal Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Partially Mimicked Development of TSC2 Neurological Abnormalities.

SummaryTuberous sclerosis complex (TSC) is a disease featuring devastating and therapeutically challenging neurological abnormalities. However, there is a lack of specific neural progenitor cell models for TSC. Here, the pathology of TSC was studied using primitive neural stem cells (pNSCs) from a patient presenting a c.1444-2A>C mutation in TSC2. We found that TSC2 pNSCs had higher proliferative activity and increased PAX6 expression compared with those of control pNSCs. Neurons differentiated from TSC2 pNSCs showed enlargement of the soma, perturbed neurite outgrowth, and abnormal connections among cells. TSC2 astrocytes had increased saturation density and higher proliferative activity. Moreover, the activity of the mTOR pathway was enhanced in pNSCs and induced in neurons and astrocytes. Thus, our results suggested that TSC2 heterozygosity caused neurological malformations in pNSCs, indicating that its heterozygosity might be sufficient for the development of neurological abnormalities in patients.