Fig 1: Nuclease activity of different Cas9-sgRNA complexes. (A) Cleavage positions of Cas9-sgRNA complexes within their respective target DNA fragments and the expected fragment sizes generated after digest are shown. (B) 200 ng of PCR-amplified target DNA were digested for 3 hr using the Cas9-sgRNA complexes shown in Figure 3, and the products then run on an agarose gel. Cas9-sgRNA-B and Cas9-sgRNA-C complexes cut their target sequence and were chosen for further experiments. Cas9-sgRNA-A and Cas9-sgRNA-D complexes failed to completely digest their target sequence. Red arrows indicate the fragments, uncut and after digestion. Lane 1 is uncut 1376-bp 5' target DNA fragment. Lane 2 is Cas9-sgRNA-A cut 5' target DNA; expected fragments are 835 and 535 bp. Lane 3 is Cas9-sgRNA-B cut 5' target DNA; expected fragments are 877 and 499 bp. Lane 4 is a marker, which is 8 µl of DNA ladder (New England, BioLabs, cat. no. N3200L). Lane 5 is uncut 1408-bp 3' target DNA fragment. Lane 6 is Cas9-sgRNA-C cut 3' target DNA; expected fragments are 975 and 433 bp. Lane 7 is Cas9-sgRNA-D cut 3' target DNA; expected fragments are 1075 and 333 bp. (C) Basic Protocol 1 results in untreated human genomic DNA from suspension cells with a size that ranges between 50 and 250 kb. To check the size of the untreated human genomic DNA and Cas9-treated DNA, CHEF electrophoresis should be run. As an example, uncut and cut commercial human genomic DNA is shown. Lane 1 is a marker that corresponds to the MidRange PFG Marker I (New England BioLabs, cat. no. N0342S). Lane 3 is 1 µg of uncut commercial human genomic DNA (Promega, cat. no. G304A) used for CRISPR-TAR cloning of the human NBS1 gene. Lane 4 is 1 µg of human genomic DNA digested overnight with 1 µl of Cas9 enzyme, 1 µl of gRNA-B, and 1 µl of gRNA-C in a total volume of 40 µl. This figure is adapted from Lee et al. (2015).