Figure 1 | Scientific Reports

Figure 1

From: CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium

Figure 1

Endogenous tRNA system for expressing sgRNAs. (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.