Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

Combi-CRISPR: combination of NHEJ and HDR provides efficient and precise plasmid-based knock-ins in mice and rats.

CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

A heterozygous LAMA5 variant may contribute to slowly progressive, vinculin-enhanced familial FSGS and pulmonary defects.

The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause a systemic developmental syndrome including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic diseases have remained unclear. We performed whole-exome sequencing analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis, and we identified what we believe to be a novel probable pathogenic variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 laminin globular 4-5 (LG4-5) domain. Heterozygous and homozygous knockin mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels, which suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Reexaminations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.

Digenic mutations in ALDH2 and ADH5 impair formaldehyde clearance and cause a multisystem disorder, AMeD syndrome.

Rs671 in the aldehyde dehydrogenase 2 gene (ALDH2) is the cause of Asian alcohol flushing response after drinking. ALDH2 detoxifies endogenous aldehydes, which are the major source of DNA damage repaired by the Fanconi anemia pathway. Here, we show that the rs671 defective allele in combination with mutations in the alcohol dehydrogenase 5 gene, which encodes formaldehyde dehydrogenase (ADH5FDH ), causes a previously unidentified disorder, AMeD (aplastic anemia, mental retardation, and dwarfism) syndrome. Cellular studies revealed that a decrease in the formaldehyde tolerance underlies a loss of differentiation and proliferation capacity of hematopoietic stem cells. Moreover, Adh5-/-Aldh2 E506K/E506K double-deficient mice recapitulated key clinical features of AMeDS, showing short life span, dwarfism, and hematopoietic failure. Collectively, our results suggest that the combined deficiency of formaldehyde clearance mechanisms leads to the complex clinical features due to overload of formaldehyde-induced DNA damage, thereby saturation of DNA repair processes.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Pitfalls in mouse norovirus (MNV) detection in fecal samples using RT-PCR, and construction of new MNV-specific primers.

The murine norovirus (MNV), which belongs to the Caliciviridae family, is prevalent in laboratory mice. Since this virus affects macrophages and dendritic cells, infected mice are not suitable for immunological investigations, making it important to detect MNV infections accurately. When we tested RNA extracts derived from mouse feces for MNV detection using nested RT-PCR with a set of MNV-specific primers reported by Goto et al. (Exp. Anim. 58: 135-140, 2009), we found that these primers amplified not only an MNV-specific signal but also amplified a relatively weak signal with a size almost identical to that of the specific signal. Analysis of the nucleotide sequence of this amplified signal revealed that it was at least 98% identical to the exophosphatase gene of a commensal bacterium, Bacteroides vulgatus. Subsequent analysis showed that the signal amplified with a pair of nested primers was from DNA derived from B. vulgatus, which is sometimes present in SPF laboratory mouse feces, and the nested primers used were both partly homologous with the B. vulgatus nucleotide sequence. We thus designed a new set of nested RT-PCR primers that was not cross-reactive with the B. vulgatus genome. PCR products amplified by the newly designed primers were at least 89.3% identical to the MNV RNA polymerase gene in all cases. Our findings demonstrated that the primer set we designed was suitable for detecting an MNV-specific signal without cross-reacting with B. vulgatus DNA in mouse feces.

A diagnostic method for pneumocystis carinii a causative agent of pneumonia in immunodeficient rats.

Immunodeficient animals are in demand in current biomedical research, and they contribute to medical progress. In Pneumocystis infections, a specific histological diagnostic tool may be immunochemistry (IC). However, it was recently reported that the antibody (3F6) was not suitable for detecting Pneumocystis in rats. We purchased another antibody [PNC007] from a commercial source for IC. We could detect positive signals at identical locations with IC and Toluidine blue O in lungs of infected rats. These results corresponded to the results obtained with PCR. We should study the relationship between unexpected positive signals seen in IC and trophic forms in lungs of infected rats. We could clinically diagnose pneumonia caused by Pneumocystis carinii with the diagnostic method we developed.