Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.

BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination. METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.

Functional and long-lived melanocytes from human pluripotent stem cells with transient ectopic expression of JMJD3.

BACKGROUND: Melanocytes are an essential part of the epidermis, and their regeneration has received much attention because propagation of human adult melanocytes in vitro is too slow for clinical use. Differentiation from human pluripotent stem cells to melanocytes has been reported, but the protocols to produce them require multiple and complex differentiation steps. METHOD: We differentiated human embryonic stem cells (hESCs) that transiently express JMJD3 to pigmented cells. We investigated whether the pigmented cells have melanocytic characteristics and functions by qRT-PCR, immunocytochemical analysis and flow cytometry. We also investigated their biocompatibility by injecting the cells into immunodeficient mice for clinical use. RESULT: We successfully differentiated and established a pure culture of melanocytes. The melanocytes maintained their growth rate for a long time, approximately 200 days, and were functional. They exhibited melanogenesis and transfer of melanin to peripheral keratinocytes. Moreover, melanocytes simulated the developmental processes from melanoblasts to melanocytes. The melanocytes had high engraftability and biocompatibility in the immunodeficient mice. CONCLUSION: The robust generation of functional and long-lived melanocytes are key to developing clinical applications for the treatment of pigmentary skin disorders.

Immediate postnatal central hypothyroidism caused by maternal Graves' disease: Importance of early screening.

This report illustrates a case of central hypothyroidism in a newborn immediately after birth caused by maternal Graves' disease. Infants from mothers with Graves' disease require careful examination without waiting for neonatal screening results, even though the mother's thyroid function is normal at birth or the newborn does not have goiter.

Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.