Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks Journal Article
| Authors: | Renouf, B.; Piganeau, M.; Ghezraoui, H.; Jasin, M.; Brunet, E. |
| Article Title: | Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks |
| Abstract: | Recurrent chromosomal translocations are found in numerous tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci. Using different PCR-based methods, we also explain how to quantify translocation frequency and to analyze breakpoint junctions in the cells of interest. In addition, PCR detection of translocations is used as a very sensitive method to detect off-target effects, which has general utility. © 2014 Elsevier Inc. |
| Keywords: | controlled study; unclassified drug; human cell; polymerase chain reaction; gene locus; wild type; rna; cancer genetics; double stranded dna; plasmid; nuclease; chromosome translocation; double stranded dna break; mesenchymal stem cell; gene induction; dna double-strand breaks; chromosomal translocations; npm-alk; ews-fli1; human; priority journal; article; cas9; cas9d10a; paired nicks ncas9; cas9 nickase; cas9 nuclease |
| Journal Title: | Methods in Enzymology |
| Volume: | 546 |
| ISSN: | 0076-6879 |
| Publisher: | Academic Press |
| Date Published: | 2014-01-01 |
| Start Page: | 251 |
| End Page: | 271 |
| Language: | English |
| DOI: | 10.1016/B978-0-12-801185-0.00012-X |
| PROVIDER: | scopus |
| PMCID: | PMC4398311 |
| PUBMED: | 25398344 |
| DOI/URL: | |
| Notes: | Chapter 12 in "The Use of CRISPR/Cas9, ZFNs, and TALENs in Generating Site-Specific Genome Alterations" (ISBN: 978-0-12-801185-0) -- Export Date: 4 May 2015 -- Source: Scopus |
