Targeted genomic rearrangements using CRISPR/Cas technology

Abstract

Genomic rearrangements are frequently observed in cancer cells but have been difficult to generate in a highly specific manner for functional analysis. Here we report the application of CRISPR/Cas technology to successfully generate several types of chromosomal rearrangements implicated as driver events in lung cancer, including the CD74-ROS1 translocation event and the EML4-ALK and KIF5B-RET inversion events. Our results demonstrate that Cas9-induced DNA breaks promote efficient rearrangement between pairs of targeted loci, providing a highly tractable approach for the study of genomic rearrangements.

Figure 1. Cas9-induced DNA breaks promote interchromosomal translocations

a) Schematic depicting the overall strategy for generating chromosomal rearrangements. Cas9 from S.pyogenes (SpCas9) is co-expressed with two single-guide RNAs (sgRNA 1 and 2) which direct DNA cleavage at each targeted genomic site. b) The CD74-ROS1 rearrangement results from a translocation between chromosomes 5 and 6. Shown are the intronic sites where Cas9 was targeted. c-d) PCR detection of the c) CD74-ROS1 Der(6) and d) ROS1-CD74 Der(5) genomic breakpoint junctions from HEK 293T cells in which Cas9 was expressed with no sgRNA (vector), CD74 sgRNA alone, ROS1 sgRNA alone, or both CD74 and ROS1 sgRNAs. e) Sequence chromatogram of the detected CD74-ROS1 fusion transcript from cells in which Cas9 and both CD74 and ROS1 sgRNAs were expressed. Data shown are representative results from a total of three independent experiments.

Figure 2. Cas9 can be targeted to generate paracentric and pericentric intrachromosomal inversions

a) The EML4-ALK rearrangement results from a paracentric inversion in chromosome 2. Shown are the intronic sites where Cas9 was targeted. b-c) PCR detection of the EML4-ALK b) genomic breakpoint junction and c) fusion transcript from HEK 293T cells in which Cas9 was expressed with no sgRNA (vector), EML4 sgRNA alone, ALK sgRNA alone, or both EML4 and ALK sgRNAs. d) Sequence chromatogram of the detected EML4-ALK fusion transcript from cells in which Cas9 and both EML4 and ALK sgRNAs were expressed. e) Western blot for EML4-ALK protein expression in single clones of 293T cells which were untreated, or in which Cas9 and both EML4 and ALK sgRNAs were expressed. NCIH3122 cells are shown as a positive control. f) The KIF5B-RET rearrangement results from a pericentric inversion in chromosome 10. Shown are the intronic sites where Cas9 was targeted. g-h) PCR detection of the KIF5B-RET g) genomic breakpoint junction and h) fusion transcript from HEK 293T cells in which Cas9 was expressed with no sgRNA (vector), KIF5B sgRNA alone, RET sgRNA alone, or both KIF5B and RET sgRNAs. i) Sequence chromatogram of the detected KIF5B-RET fusion transcript from cells in which Cas9 and both KIF5B and RET sgRNAs were expressed. Data shown are representative results from a total of three independent experiments.

Figure 3. Estimated efficiencies of inducing EML4-ALK and KIF5B-RET rearrangements

HEK 293T cells transfected with Cas9 and no sgRNA (Vector), a single sgRNA, or a pair of sgRNAs were stained for either (a) ALK or (b) RET protein to estimate the percentage of cells with induced EML4-ALK or KIF5B-RET rearrangements, respectively. All values represent means of at least 3 independent experiments +/− s.e.m. Means were compared by paired t-test (**p<0.01, ***p<0.001). Also shown are dotplots from a representative experiment for each rearrangement.