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Download ToolGen Presentation - The National Academies of Sciences
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Genome Editing: Challenges and Opportunities Jin-Soo Kim Dept. of Chemistry, Seoul National Univ. Center for Genome Engineering, Institute for Basic Science 1 Challenges in Genome Editing • Delivery • Immunogenicity • Mosaicism • HDR vs. NHEJ • Specificity: Off-target mutations • Regulatory guidelines 2 Genome Surgery Plasmid Ex vivo therapy In vivo therapy Cationic lipid mRNA + gRNA RNP AAV Adenovirus Viral vector www.toolgen.com 3 • T cells from HIV+ patients are treated with a programmable nuclease. • CCR5-inactive T cells are delivered back to patients 4 Stem Cell Therapy: Gene Correction in iPS Cells Patient’s somatic cells Reprogramming iPS cells Biopsy Gene correction Patient Transplantation Differentiation Gene-corrected iPS cells Gene-corrected cells Park et al. Cell Stem Cell (2015) 5 Nuclease Immunogenicity • Programmable nucleases are potential immunogens • In vivo delivery and long-term expression can elicit immune responses • Transient delivery of Cas9 RNPs or ex vivo delivery can be a solution 6 Double-Strand Break Repair Kim H & Kim JS, Nat. Rev. Genet. (2014) • Error-prone NHEJ is useful for targeted gene knockout • Homology-directed repair is needed to correct genetic defects • How to suppress NHEJ and enhance HDR? 7 Nuclease Off-target Effects • ZFNs, TALENs, and CRISPR-Cas9 can cleave off-target sites • Off-target mutations can - Inactivate essential genes - Activate oncogenes - Cause chromosomal rearrangements Wu et al. Quant. Biol. (2014) Off-target Chromosomal Rearrangements 3 1 4 4 1 2 3 1 Deletion 4 3 2 2 1 4 Inversion 1 2 3 4 1 1 2 3 Translocation Inversion 2 3 4 4 1 2 3 4 1 2 3 3 4 Duplication 9 How to Assess Genome-wide Off-target Effects • Whole genome sequencing: Limited by sequencing depth • Digenome-seq: Nuclease-digested whole genome seq. • Cell-based methods: GUIDE-seq, Translocation seq., BLESS Kim et al. Nat. Methods (2015) Gabriel et al. Nat. Biotechnol. (2015) How to Avoid Off-target Effects • Choose a unique target site • Use purified Cas9 protein rather than Cas9 plasmid • Use modified guide RNAs • Use modified Cas9: paired Cas9 nickases, Cas9-ZFP, dCas9-FokI Koo et al. Molecules and Cells (2015) Do Off-target Effects Matter? • No drugs are free from off-target effects, often leading to repositioning • Etoposide, an anti-cancer drug, cleaves DNA randomly, inducing mutations • Biological consequences rather than mutations per se are more relevant • CCR5-targeted ZFN has been proven safe in a clinical test (thus far)
