Wild-type Cas9 (upper) and nickase-type Cas9 (bottom). This is a schematic representation of DNA double digestion by wild type Cas9 and Cas9 D10A nickases (Cas9n). The D10A mutation renders Cas9 able to cleave only the strand complementary to the sgRNA; a pair of sgRNA-Cas9n complexes can nick both strands simultaneously. Compared to conventional methods that are only recognized by 20 nucleotides, in this double-cincture method, two sgRNAs recognize a target site of 40 (20 + 20) nucleotides for higher specificity.