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![Page 1: 1 Jin-Soo Kim Dept. of Chemistry, Seoul National Univ. Center for Genome Engineering, Institute for Basic Science Genome Editing: Challenges and Opportunities.](https://reader035.fdocuments.us/reader035/viewer/2022070413/5697bf9b1a28abf838c92f6c/html5/thumbnails/1.jpg)
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Jin-Soo KimDept. of Chemistry, Seoul National Univ.Center for Genome Engineering, Institute for Basic Science
Genome Editing: Challenges and Opportunities
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Challenges in Genome Editing
• Delivery
• Immunogenicity
• Mosaicism
• HDR vs. NHEJ
• Specificity: Off-target mutations
• Regulatory guidelines
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www.toolgen.com 3
Genome Surgery
AAV
Cationic lipid
Ex vivo therapy In vivo therapyPlasmid
mRNA + gRNA
RNP
Viral vector Adenovirus
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• T cells from HIV+ patients are treated with a programmable nuclease.
• CCR5-inactive T cells are delivered back to patients
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Stem Cell Therapy: Gene Correction in iPS Cells
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Patient’s somatic cells
iPS cells
Gene-corrected iPS cells
Gene-corrected cells
Differentiation
Gene correction
Reprogramming
Biopsy
Transplantation
Patient
Park et al. Cell Stem Cell (2015)
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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
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Double-Strand Break Repair
• 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?
Kim H & Kim JS, Nat. Rev. Genet. (2014)
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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)
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Off-target Chromosomal Rearrangements
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Deletion
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Inversion
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Translocation
Inversion
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Duplication
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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
Gabriel et al. Nat. Biotechnol. (2015)Kim et al. Nat. Methods (2015)
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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)
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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)