DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.     

Identification of major proteins in maize egg cells

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.     

A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier

Several studies have addressed the importance of various ubiquitin-like (UBL) post-translational modifiers. These UBLs are covalently linked to most, if not all, target protein(s) through an enzymatic cascade analogous to ubiquitylation, consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. In this report, we describe the identification of a novel ubiquitin-fold modifier 1 (Ufm1) with a molecular mass of 9.1 kDa, displaying apparently similar tertiary structure, although lacking obvious sequence identity, to ubiquitin. Ufm1 is first cleaved at the C-terminus to expose its conserved Gly residue. This Gly residue is essential for its subsequent conjugating reactions. The C-terminally processed Ufm1 is activated by a novel E1-like enzyme, Uba5, by forming a high-energy thioester bond. Activated Ufm1 is then transferred to its cognate E2-like enzyme, Ufc1, in a similar thioester linkage. Ufm1 forms several complexes in HEK293 cells and mouse tissues, revealing that it conjugates to the target proteins. Ufm1, Uba5, and Ufc1 are all conserved in metazoa and plants but not in yeast, suggesting its potential roles in various multicellular organisms.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

TetraThymosinbeta is required for actin dynamics in Caenorhabditis elegans and acts via functionally different actin-binding repeats

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats, each related to the actin monomer-sequestering protein thymosinbeta 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Preparation of recombinant rat eosinophil-associated ribonuclease-1 and -2 and analysis of their biological activities

Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.