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Collectively, our results indicate that both an appropriate LET value and an adequate particle number might be needed to obtain the highest mutation efficiency with heavy-ion beam irradiation.
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C ions at LETmax showed higher mutation efficiency than those with LET of 22.5 keV μm-1, with an efficiency that appears similar to that with EMS.
The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations.
Overall, caution is warranted when performing validation trials with dideoxy-sequencing as the golden standard to determine the specificity of methods with higher mutation detection efficiency on FFPE-DNA templates.
However, the observed mutation efficiency should be high enough to generate heritable mutations according to our experience in silkworm.
This mutation efficiency is ten-fold higher than the 3-7 % obtained with transcription-activator like effector nucleases (TALENs) in soybean hairy roots [ 30].
A higher copy number in the donor transposon concatemer may increase mutation efficiency due to more "jumping" transposons in the sperm of NOD seed males (Geurts et al. 2006).
The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site.
Therefore, mutation efficiency is easily underestimated.
Here, we describe methods for constructing TALEN vectors that have been shown to achieve high success rates and mutation efficiencies in zebrafish.
Interestingly, in these cloned embryos, we found high point mutation repairing efficiency, which was between 7.0% and 25.9% among the repaired embryos (Figs. 3C, S5 and Table S1).
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