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Fig. 3 | BMC Biotechnology

Fig. 3

From: Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

Fig. 3

The influence of 5′ sgRNA supernumerary GG residues on cleavage efficiencies at targets with or without matching 5′ GG in BmGR66. A T7EN1 assay was performed after GR66-T5 was targeted by sgRNAs in the GGN18 and GGN20 formats. Cleavage products were separated by PAGE. Wildtype (WT) controls were amplified from Dazao genomic DNA by PCR. B Cleavage efficiencies were calculated by measuring band intensities. Bars represent mean values, and error bars represent SEM. P-values were determined using Student’s t-test. NS, not significant (P > 0.05). C A T7EN1 assay was performed to examine the cleavage efficiencies of sgRNAs targeting T1, T2, T3, and T4. WT-T1 (500 bp) was the uncleaved control for sgRNA GR66-T1, and WT-T234 (565 bp) was the uncleaved control for sgRNAs GR66-T2, T3, and T4. The two bands derived from T7EN1 digestion for sgRNA T1 were 248 bp and 252 bp; for sgRNA T2, 262 bp and 303 bp; for sgRNA T3, 274 bp and 291 bp; for sgRNA T4, 238 bp and 327 bp. Wildtype (WT) controls were amplified from Dazao genomic DNA by PCR. Cleavage efficiencies were calculated by measuring band intensities. Efficiency values are shown under each lane. Only one band was detected for sgRNA T1, because the two bands derived from T7EN1 cleavage are almost identical in size and were not resolved by the PAGE conditions used. Please note that the uncropped original PAGE gel images are shown in Additional file 1: Figure S3

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