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Oral: The Function and Mechanism of CRISPR and Its Applications


CRISPR RNA-guided Genome Editing in Human Stem Cells, Animals, and Plants
J. KIM (1) (1) Center for Genome Engineering, Institute for Basic Science, South Korea

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Genome editing that allows targeted mutagenesis in higher eukaryotic cells and organisms is broadly useful in biology, biotechnology, and medicine. We have developed ZFNs, TALENs, and Cas9 RNA-guided endonucleases (RGENs), derived from the type II CRISPR-Cas prokaryotic adaptive immune system, to modify chromosomal DNA in a targeted manner. In particular, we used purified Cas9 protein and in vitro transcribed guide RNAs rather than plasmids encoding these components to correct large chromosomal inversions in the blood coagulation factor VIII gene that cause hemophilia A in patient-derived induced pluripotent stem cells (iPSCs) and to modify diverse genes in large animals and plants. The resulting animals and plants contained small insertions or deletions (indels) at target sites, which are indistinguishable from naturally-occurring variations, possibly bypassing regulatory requirements associated with use of recombinant DNA. Despite broad interest in RNA-guided genome editing, RGENs are limited by off-target mutations. We developed Cas9 nuclease-digested whole genome (digenome) sequencing (Digenome-seq) to profile genome-wide specificities of Cas9 nucleases in an unbiased manner. Digenome-seq captured nuclease cleavage sites at single nucleotide resolution and identified off-target sites at which indels were induced with frequencies below 0.1%.