Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.
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.
Museum National d'Histoire Naturelle, INSERM U1154, CNRS 7196, Paris, France.
Benjamin
B
Renouf
Museum National d'Histoire Naturelle, INSERM U1154, CNRS 7196, Paris, France.
Marion
M
Piganeau
Museum National d'Histoire Naturelle, INSERM U1154, CNRS 7196, Paris, France.
Hind
H
Ghezraoui
Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA. Electronic address: ebrunet@mnhn.fr.
Maria
M
Jasin
Museum National d'Histoire Naturelle, INSERM U1154, CNRS 7196, Paris, France. Electronic address: m-jasin@ski.mskcc.org.
Erika
E
Brunet
D001483:Base Sequence; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods; D014178:Translocation, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems
D003850:Deoxyribonuclease I; D000094704:RNA, Guide, CRISPR-Cas Systems
Cas9; Cas9D10A; Chromosomal translocations; DNA double-strand breaks; EWS–FLI1; NPM–ALK; Paired nicks; nCas9