CRISPR Genome Editing

Genome editing with CRISPR systems that allows targeted mutagenesis in cells and organisms is broadly useful in biology, biotechnology, and medicine. Despite broad interest in CRISPR RNA-guided genome editing, Cas9, Cpf1, and Cas9-fused deaminases (a.k.a., Base Editors) are limited by off-target mutations. We developed nuclease-digested whole genome sequencing (Digenome-seq) to profile genome-wide specificities of Cas9 and Cpf1 nucleases and Cas9-fused deaminases in an unbiased manner. Digenome-seq captured in vitro cleavage sites at single nucleotide resolution and identified off-target sites at which indels or base conversions were induced with frequencies below 0.1%. We also showed that these off-target effects could be avoided by using preassembled ribonucleoproteins (RNPs) and modified guide RNAs. Digenome-seq is a robust, sensitive, unbiased, and cost-effective (< USD 1,500) method for profiling genome-wide off-target effects of programmable nucleases and deaminases.

20180524_세미나_김진수 교수.pdf