[BME Colloquium] Genome Editing in Cultured Cells and Whole Organisms with Programmable Nucleases
  • Jin-Soo Kim, Ph.D. (Seoul National University)
  • SLS Colloquia / Sep 18th 2014 04:00 pm / EB1 E104
Genome editing that allows targeted mutagenesis in higher eukaryotic cells and organisms is broadly useful in biology, biotechnology, and medicine. We have developed zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Cas9 RNA-guided engineered nucleases (RGENs), derived from the type II CRISPR/Cas prokaryotic adaptive immune system, to cleave chromosomal DNA in a targeted manner, producing DNA double-strand breaks in cells, the repair of which via endogenous systems gives rise to targeted genome modifications. The Cas9 protein, when complexed with small guide RNAs (sgRNAs), recognizes and cleaves target DNA sequences complementary to the guide RNAs in vivo, inducing targeted genome modifications at high frequencies in cultured cells and whole organisms. Unlike ZFNs and TALENs, RGENs can be customized without any cloning step, making them a broadly useful, scalable and expeditious platform for genome editing. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. Furthermore, we deliver purified recombinant Cas9 protein complexed with sgRNAs (RGEN ribonucleoproteins (RNPs)) to animal embryos and cultured human cells including hard-to-transfect pluripotent stem cells to achieve highly efficient RNA-guided genome editing in cells and whole organisms. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects and mosaicism.