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. Author manuscript; available in PMC: 2015 Apr 15.
Published in final edited form as: Methods Enzymol. 2014;546:251–271. doi: 10.1016/B978-0-12-801185-0.00012-X

Figure 1. Induction of the NPM-ALK translocation by Cas9 DSBs or nCas9 paired nicks.

Figure 1

A. Induction of DSBs or paired nicks at the NPM and ALK loci can lead to chromosomal translocations and expression of the NPM-ALK fusion protein from Der5.

B. Position of the gRNAs. With wild-type Cas9, DSBs are formed using gRNAs NPM1 and ALK1. With nCas9, paired nicks are formed using NPM1+NPM2 (37 bp apart) and ALK1+ALK2 (41 bp apart), resulting in 5′ overhangs if the strands are separated.

C. Translocations are only detected when two DSBs or two paired nicks are introduced. PCR is performed with primers indicated in A.

D. Estimate of cleavage efficiency at the ALK locus using wild-type Cas9 and either the ALK1 or ALK2 gRNA and nCas9 with both the ALK1 and ALK2 gRNAs. The T7 assay detects 60% indel formation in all three instances.