Base-pairing of sgRNAs in the seed-region blocks Cas9-mediated cleavage of a test-plasmid. (A,B) Structure of sgRNAs. Horizontal red lines in A and B indicate artificially introduced base-pairing with the sgRNA scaffold, and vertical red lines in a indicate potential base-pairing with nucleotides in loop 2. Nucleotides base-pairing in loop 1 are bold. (C) Denaturing acrylamide gel showing synthetic oligonucleotides before and after sgRNA/Cas9-mediated cleavage for 1 h. Note that Cas9 cleavage is heterogeneous. (D) 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.
