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.

Evolution of new protein topologies through multistep gene rearrangements.

New protein folds have emerged throughout evolution, but it remains unclear how a protein fold can evolve while maintaining its function, particularly when fold changes require several sequential gene rearrangements. Here, we explored hypothetical evolutionary pathways linking different topological families of the DNA-methyltransferase superfamily. These pathways entail successive gene rearrangements through a series of intermediates, all of which should be sufficiently active to maintain the organism's fitness. By means of directed evolution, and starting from HaeIII methyltransferase (M.HaeIII), we selected all the required intermediates along these paths (a duplicated fused gene and duplicates partially truncated at their 5' or 3' coding regions) that maintained function in vivo. These intermediates led to new functional genes that resembled natural methyltransferases from three known classes or that belonged to a new class first seen in our evolution experiments and subsequently identified in natural genomes. Our findings show that new protein topologies can evolve gradually through multistep gene rearrangements and provide new insights regarding these processes.

The formation of Escherichia coli curli amyloid fibrils is mediated by prion-like peptide repeats.

Amyloid fibril formation is the hallmark of major human maladies including Alzheimer's disease, type II diabetes, and prion diseases. Prion-like phenomena were also observed in yeast. Although not evolutionarily related, one similarity between the animal PrP and the yeast Sup35 prion proteins is the occurrence of short peptide repeats that are assumed to play a key role in the assembly of the amyloid structures. It was recently demonstrated that typical amyloid fibril formation is associated with biofilm formation by Escherichia coli. Here, we note the functional and structural similarity between oligopeptide repeats of the major curli protein and those of animal and yeast prions. We demonstrate that synthetic peptides corresponding to the repeats form fibrillar structures. Furthermore, conjugation of beta-breaker elements to the prion-like repeat significantly inhibits amyloid formation and cell invasion of curli-expressing bacteria. This implies a functional role of the repeat in the self-assembly of the fibrils. Since mammal prion, yeast prion, and curli protein are evolutionarily distinct, the conserved peptide repeats most likely define an optimized self-association motif that was independently evolved by diverse systems.

The YoeB toxin is a folded protein that forms a physical complex with the unfolded YefM antitoxin. Implications for a structural-based differential stability of toxin-antitoxin systems.

The chromosomal YoeB-YefM toxin-antitoxin module common to numerous strains of bacteria is presumed to have a significant role in survival under stringent conditions. Recently we showed that the purified YefM antitoxin is a natively unfolded protein, as we previously reported for the Phd antitoxin in the P1 phage Doc-Phd toxin-antitoxin system. Here we report the purification and structural properties of the YoeB toxin and present physical evidence for the existence of a tight YoeB. YefM polypeptide complex in solution. YoeB and YefM proteins co-eluted as single peaks in sequential Ni-affinity FPLC and Q-Sepharose ion-exchange chromatography implying the formation of a YoeB. YefM complex. The unstable antitoxin was removed from the mixture by natural proteolysis, and the residual YoeB protein was purified using ion exchange chromatography. Fluorescence anisotropy studies of the purified YoeB and YefM proteins showed a 2:1 stoichiometry of the complex, providing direct evidence for a physical complex between the proteins. Near- and far-UV circular dichroism spectroscopy of the purified toxin revealed that, similar to the Doc toxin, YoeB is a well-folded protein. Thermal denaturation experiments confirmed the conformational stability of the YoeB toxin, which underwent reversible thermal unfolding at temperatures up to 56 degrees C. The thermodynamic features of the toxin-antitoxin complex were similar. Taken together, our results support the notion of a correlation between differential physiological and structural stability in toxin-antitoxin modules.