Engineering genomic deletions and inversions in mouse ES cells using custom designed nucleases (Prot 62)
The recent development of custom designed nucleases, such as Zinc-Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeat Associated system (CRISPR/Cas9) has opened up exciting opportunities to edit genomes in a wide range of organisms (Joung and Sander, 2013 for review). Knocking out protein-coding genes can be easily achieved by using just one pair of such dimeric nucleases, to target the first coding exon, thereby introducing short indels that result in a translational frameshift. Several reports have also demonstrated the possibility to target larger genomic rearrangements by using two pairs of nucleases (Carlson et al., 2012; Gupta et al., 2013; Lee et al., 2011). Although homologous recombination mediated genetic engineering is feasible in some systems, such as mouse embryonic stem cells, this approach requires multiple steps, including the selection of drug-resistant clones, and can be laborious depending on the target and nature of the targeting. This approach is being rapidly superceded by the advent of custom ZFN, TALEN and CRISPR/Cas9 technologies, which enable the extremely rapid and efficient disruption of not only coding, but also non-coding elements, by creating deletions, or by changing local genomic organization by creating inversions.
Institut Curie, 26 rue d’Ulm 75005 Paris France
Corresponding author: Elphège P Nora, Edith Heard
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