The secondary structure of sgRNAs affects Cas9-mediated cleavage efficiency. (A) Sequences of sgRNAs. Small letter guanosines used for in vitro transcription are not present in the target DNA sequence. (B) Agarose gel showing Cas9-mediated cleavage after 6 h of 3 kb pBS SK+ test-plasmids containing the target sequence with indicated sgRNAs. Plasmids were linearized with ScaI as in the control after Cas9 heat inactivation. Size markers are EcoRI/HindIII digested λ DNA of 20 kb, 3.6 kb, 1.9 kb, and 0.8 kb. (C) Denaturing acrylamide gel showing synthetic oligonucleotides before and after sgRNA/Cas9-mediated cleavage for 1 h. (D–G) Structure of sgRNAs. Nucleotides base-pairing in loop 1 are bold. Red lines in D indicate potential base-pairing with nucleotides in loop 2. Green nucleotides indicate mutations compared to sgRNA R13G.
