Gene editing on-/off-target analyses

Genome editing is based on designer nucleases (Zinc Finger Nucleases, ZFN; Transcription Activator-Like Effector Nuclease, TALEN; Clustered Regularly Interspaced Short Palindromic Repeats / Cas, CRISPR/Cas; Meganucleases) that allow the targeted introduction of genetic material into a specific locus of the mammalian genome. Genome editing has reached convincing and promising results in basic research and clinical studies. However, concerns remain on the possibility that unknown off-target mutations may cause malignant transformation, because in vivo cleavage sites and introduced DNA damage cannot be comprehensively predicted in silico, requiring practical studies (Gabriel et al. Nat Biotech 2011).

Off-target analyses based on (nr)LAM-PCR or target enrichment sequencing may be performed using the sequence information of the target genomic locus or of the applied donor sequence (e.g. plasmid, viral vectors) (Gabriel et al. Nat Biotech 2015). Bioinformatically defined off-targets are analyzed by locus-specific PCR based NGS.

References

  1. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nature Biotechnology. 29, 816-823, 2011.
  2. Gabriel R, von Kalle C, Schmidt M. Mapping the precision of genome editing. Nature Biotechnology. 33, 150-152, 2015.
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