Figure 2. CATCH cleavage and cloning of long genomic sequences of various lengths.

(a) A total of six sgRNA pairs were designed to target genome segments of different lengths (50, 75, 100, 150 and 200 kb, respectively, including one pair targeting 50 kb but with the PAM sequences falling on the flanking sequences instead of the target genome segment) in the E. coli str. K-12 substr. MG1655 genome, all containing a lacZ gene. The genome location, target sequences, PAM sequences and cleavage sites are detailed and highlighted. (b) E. coli chromosomes in agarose gel plugs were digested by Cas9 with the corresponding sgRNA pairs and analysed using PFGE. (c) A 50-kb segment on the E. coli chromosomes was targeted by two sgRNA pairs of opposite orientations, and the cleavage products were analysed using PFGE. M, marker.