Mutant allele knockout with novel CRISPR nuclease promotes myelopoiesis in ELANE neutropenia.

Severe congenital neutropenia (SCN) is a life-threatening marrow failure disorder, usually caused by heterozygous mutations in ELANE. Potential genetic treatment strategies include biallelic knockout or gene correction via homology-directed repair (HDR). Such strategies, however, involve the potential loss of the essential function of the normal allele product or limited coverage of diverse monogenic mutations within the patient population, respectively. As an alternative, we have developed a novel CRISPR-based monoallelic knockout strategy that precisely targets the heterozygous sites of single-nucleotide polymorphisms (SNPs) associated with most ELANE mutated alleles. In vitro studies demonstrate that patients' unedited hematopoietic CD34+ cells have significant abnormalities in differentiation and maturation, consistent with the hematopoietic defect in SCN patients. Selective knockout of the mutant ELANE allele alleviated these cellular abnormalities and resulted in about 50%-70% increase in normally functioning neutrophils (p < 0.0001). Genomic analysis confirmed that ELANE knockout was specific to the mutant allele and involved no off-targets. These results demonstrate the therapeutic potential of selective allele editing that may be applicable to SCN and other autosomal dominant disorders.

Computational Design of Epitope-Specific Functional Antibodies.

The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.

New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order.

Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a ß-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and (1)H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.

An engineered microbial platform for direct biofuel production from brown macroalgae.

Prospecting macroalgae (seaweeds) as feedstocks for bioconversion into biofuels and commodity chemical compounds is limited primarily by the availability of tractable microorganisms that can metabolize alginate polysaccharides. Here, we present the discovery of a 36-kilo-base pair DNA fragment from Vibrio splendidus encoding enzymes for alginate transport and metabolism. The genomic integration of this ensemble, together with an engineered system for extracellular alginate depolymerization, generated a microbial platform that can simultaneously degrade, uptake, and metabolize alginate. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).

Genetic constraints on protein evolution.

Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.

Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries.

The directed evolution of proteins has benefited greatly from site-specific methods of diversification such as saturation mutagenesis. These techniques target diversity to a number of chosen positions that are usually non-contiguous in the protein's primary structure. However, the number of targeted positions can be large, thus leading to impractically large library size, wherein almost all library variants are inactive and the likelihood of selecting desirable properties is extremely small. We describe a versatile combinatorial method for the partial diversification of large sets of residues. Our library oligonucleotides comprise randomized codons that are flanked by wild-type sequences. Adding these oligonucleotides to an assembly PCR of wild-type gene fragments incorporates the randomized cassettes, at their target sites, into the reassembled gene. Varying the oligonucleotides concentration resulted in library variants that carry a different average number of mutated positions that comprise a random subset of the entire set of diversified codons. This method, dubbed Incorporating Synthetic Oligos via Gene Reassembly (ISOR), was used to create libraries of a cytosine-C5 methyltransferase wherein 45 individual positions were randomized. One library, containing an average of 5.6 mutated residues per gene, was selected, and mutants with wild-type-like activities isolated. We also created libraries of serum paraoxonase PON1 harboring insertions and deletions (indels) in various areas surrounding the active site. Screening these libraries yielded a range of mutants with altered substrate specificities and indicated that certain regions of this enzyme have a surprisingly high tolerance to indels.

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus.

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.

Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins.

To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.