Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks. Brinkman, E.K. et al
Figure 1: We established a clonal K562 cell line with a stably integrated construct that encodes a tightly controlled inducible Cas9 nuclease. Western blot analysis of Cas9 at various time points after induction of Cas9 by Shield-1. It shows that Cas9 accumulates gradually after induction. Figure 3, 5, S3, S6: We established a variant of the ligation-mediated PCR assay for the quantification of DNA breaks at a defined location (Chailleux et al., 2014; Dai et al., 2000; Garrity and Wold, 1992). For the detection of broken DNA, we first denature the DNA and subject it to a primer extension reaction using a primer near the break site. This ensures that all cleavage sites are converted into blunt ends, even if resection of the broken ends has occurred. Next, an adaptor is ligated to the blunted DNA end, followed by PCR with one primer near the break site and a second primer that is complementary to the adaptor sequence. When analyzed on agarose gels, the samples from cells treated with sgRNA and Shield-1 yielded a band of the expected size. Figure 6: in vitro digestion by CRISPR. By PCR we first produced double-stranded DNA fragments of 600 – 1,000 bp of 3 loci in the genome. We then incubated each fragment in vitro with the respective Cas9/sgRNA complex to induce DSBs, and investigated the reaction products by agarose electrophoresis. Some samples Cas9 was subsequently denatured by two different heat treatment protocols (20 min 80 ˚C or 5 min 96 ˚C). DNA was incubated for either 2 hours or overnight.