| 研究実績の概要 |
In 2016, we evaluated the sgRNA knockdown efficiency for Ube3a using the NEURO2A cell culture. Compared with previous single-dose sgRNA targeting at the SNORD116 region, combination of sgRNAs specific SNODR116 and intron region of Ube3a have positive effects in up-regulating the Ube3A. We then tested on the primary neutron collected from the Angelman syndrome mouse which has a missing exon. We measured the expression of paternal Ube3A upon the transfection of candidate sgRNA vectors. However, the rescue of the paternal allele could not be observed. We reason that this may be related with the chromatin status of the imprinted paternal genomic region which is not accessible for the sgRNA. We suggest the sgRNA should be positioned at the open chromatin region in order to reactivate the paternal Ube3A. Following this hint, we also tried to understand the relationship between sequence features of sgRNAs and their on-target efficiencies. The lack of such insight is largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. Using the sgRNA library, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. we also demonstrated that the genomic context of the targeted DNA, the GC percentage, the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. this analysis reveals important parameters for the design of sgRNAs with high on-target efficiencies
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