Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 56 clones. Furthermore, varying concentrations of hygromycin B affected the sub-nuclear localization of dCas9-GFP, with higher concentrations leading to more uniform fluorescence distribution. These findings demonstrate that tRNA systems can effectively be employed to regulate sgRNA expression and ensure precise dCas9 targeting within the nucleus, paving the way for more efficient CRISPR/Cas9 applications in genetic engineering and research. Figure 1 Endogenous tRNA system for expressing sgRNAs (Adopted from Sekine et al., 2018) Image capton: (A) Comparison of sgRNA expression using different promoters. RT-PCR of sgRNA is presented. The lower panel reveals Ig7 as an internal control. Gel images were cropped, but no other bands were present. (B) Predicted secondary structure of tRNA-sgRNA. The nucleotides of isoleucine tRNA are indicated in red, and the DNA matching region is presented in blue. The green arrowhead indicates the tRNA cleavage site to release sgRNA. (C) Sequence analysis of the tRNA-sgRNA junction by cRT-PCR. Sequences of four independent clones are presented. As empty sgRNA vector was sequenced, the DNA matching region shown in blue contains two BpiI sites (underlined) to insert a pair of annealed oligonucleotides. The extra nucleotide at 5′ region is given in red. (D) The sub-nuclear localisation of dCas9-GFP. All the dCas9-expressing transformants were maintained with 60 µg/ml G418 for a few days before imaging. Cells expressing sgRNA were cultured with hygromycin B at 50, 100 and 200 µg/ml, respectively. Graphs reveal a histogram of standard deviation (SD) of fluorescence distribution within the nucleus. Lower SD indicates a uniform distribution of dCas9-GFP in the nucleus (Adopted from Sekine et al., 2018)
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