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SELECTBIO Conferences Genome Engineering


Profiling Genome-wide Target Specificities of CRISPR/Cas9 Nucleases in Human Cells

Jin-Soo Kim, Director/Associate Professor, Seoul National University

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 engineered nucleases (RGENs), derived from the type II CRISPR/Cas prokaryotic adaptive immune system, to cleave chromosomal DNA in a targeted manner and to edit genome sequences in human pluripotent stem cells, animals, and plants. 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. 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 to achieve highly efficient RNA-guided genome editing. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects and mosaicism. In addition, we present RGEN-digested whole genome sequencing (Digenome-seq) to profile genome-wide Cas9 off-target effects in human cells. We show that RGENs can be highly specific, inducing off-target mutations at just several sites in the entire genome and that off-target effects can be avoided by replacing these ‘promiscuous’ sgRNAs with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased, and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including RGENs.

Add to Calendar ▼2015-10-07 00:00:002015-10-08 00:00:00Europe/LondonGenome EngineeringGenome Engineering in Hanover, GermanyHanover,