New Tool for Genome Surgeryffffffff-b34e-2810... · • CRISPR system can be heterologously...
Transcript of New Tool for Genome Surgeryffffffff-b34e-2810... · • CRISPR system can be heterologously...
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New Tool for Genome Surgery
Presented by Duo Li
12.03.2013
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Location of Various Genetic Disesases
Griffiths AJF, et al.
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Potential Genome Editing
John van der Oost.
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Restriction Nuclease
• Recognize sites very shot, 4-8 bp.
• Occur too frequently in the genome.
John van der Oost.
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Homing Endonuclease• Microbial DNA-cleaving enzymes mobilize
their own reading frames by generating double strand breaks at specific genomic invasion sites.
• Recognize longer targets, 20-30bp.
• Wide range of fidelity influenced by host constraints on the coding sequence of the target gene.
• Results in the insertion, deletion, mutation or correction of DNA sequences.
• Specificity require laborious protein design.
John van der Oost.
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Homing Endonuclease and Genetic Homing
Barry L. S.
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Homing Endonuclease Structural Families
Recognition mechanisms:• Form highly elongated protein folds with minimal hydrophobic cores.• Multimerize and thereby double their DNA-contact surface Barry L. S.
• Antiparallel β-sheets,• Major grooves,• Sequence-specific and
non-specific contacts,• Distributed
nonuniformly across multiple consecutive basepairs.
• Less specific,• More heterogeneous
collection of DNA contacts,
• Major and minor groove of their target sites,
• Extended, multi-domain protein structures contact a series of DNA regions.
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Zinc Finger Nuclease
• Consists of a nuclease fused to a set of zinc finger domains interact with 3 nucleotides.
• Target up to 36 bp.
• Specificity depends on the established triplet domain.
• Modification includes: gene disruption, gene correction, targeted gene addition.
John van der Oost.
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Structure of Zinc Finger Nucleases
Fyodor D. U. et al.
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Fyodor D. U. et al.
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1 Modular Assembly
Fyodor D. U. et al.
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2 OPEN Method for Engineering Zinc-Finger Arrays
Morgan L.M. et al.
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3 Bacterial One-hybrid System
Xiangdong M. et al.
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4 Two-finger Modules
Michael M. et al.
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Types of genome editing made possible using zinc finger nucleases
Fyodor D. U. et al.
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Transcription Activator-like Effector Nuclease ( TALEN )
• Found in plant pathogenic bacteria , injected into plant cells via bacterial type III secretion system, imported into the plant cell nucleus, targeted to effector-specific gene promoters.
• A nuclease fused to a protein consisting 12-26 domains, each interact with a single base.
• Comprising tandem, polymorphic amino acid repeats.
John van der Oost, Jens B. et al.
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TAL Effector DNA Recognition
• Specificity depends on a variable number of imperfect, typically 34, aminoacid repeats.
• Polymorphism is primarily at repeat positions 12 and 13,whichwe call therepeat-variable di-residue (RVD).
• One RVD to one nucleotide, with some degeneracy and no apparentcontext dependence.
Adam J. B. et al.
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Genomic Control Enabled by Engineered TAL Effector Proteins
Adam J. B. et al.
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• Use oligonucleotides as a recognition module.
• Triple helix forming oligonucleotide conjugated to a non-specific nucleases or ‘chemical’ nucleases or type II Rease.
• DNA fragments with strands composed of either purines or pyrimidines.
• Bipartite recognition: site of the REase and the DNA sequence matching to the TFO.
Triplex-helix-forming Oligonucleotides ( TFO) with A Single-chain Nuclease
John van der Oost.
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Generate a Programmed Restriction Enzyme
TFS:triple-helix forming site.
M: 5-methyl-20-deoxycytidineP: 5-[1-propynyl]-20-deoxyuridine
one helical turn
Kristin E. et al.
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Clustered Regularly interspaced Short Palindromic Repeats ( CRISPR)
John van der Oost., and Michael P. T. et al.
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Clustered Regularly interspaced Short Palindromic Repeats ( CRISPR)
John van der Oost.
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Clustered Regularly interspaced Short Palindromic Repeats ( CRISPR)
John van der Oost
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Clustered Regularly interspaced Short Palindromic Repeats ( CRISPR)
John van der Oost
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Clustered Regularly interspaced Short Palindromic Repeats ( CRISPR)
John van der Oost
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Clustered Regularly interspaced Short Palindromic Repeats ( CRISPR)
John van der Oost
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Diversity of CRISPR-mediated Adaptive Immune Systems
Blake W. et al.
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Type I, II AND III CRPSPR Expression and Interference Stages
Edze R.W. et al.
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Edze R.W. et al.
Protospacer-adjacent Motif (PAM) Sequences Identified for CRISPR/Cas Subtypes
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Hypothetical Mechanism of CRISPR Adaptation
Edze R.W. et al.
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Cascade and Cascade-mediated R-loop Formation
Edze R.W. et al.Cascade: CRISPR-associated complex for antiviral defence
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CRISPR Interference by Type I-E System
Edze R.W. et al.
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• In Streptococcus pyogenes, identify tracrRNA
• tracrRNA directs the maturation of crRNAsby the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein.
• crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introducedouble-stranded (ds) breaks in target DNA.
• The Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand.
• The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNAcleavage.
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Type II CRISPR-mediated DNA Double-strand Break
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Optimization:S. pyogenes Cas9 (SpCas9), RNase III and tracrRNA
Comparison of different tracrRNA transcripts for Cas9-mediated gene targeting
SpCas9 and SpRNase III with NLSs enables import into the mammalian
nucleus
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Schematic Representation of Base Pairing betweenTarget Locus and EMX1- targeting crRNA
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Define a Minimal Three-component Systemfor Efficient RNA-guided Genome Modification
in Mammalian Cells293FT cells
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SpCas9 Can Be Reprogrammed to Target Multiple Genomic Loci in Mammalian Cells
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Effects of Spacer-protospacer Mismatches
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Comparison of The Efficiency of TALEN and SpCas9
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Cas9 for Homologous Recombination
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Multiplex Genome Engineering
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Protospacer Sequences and Modification Efficiencies of Mammalian Genomic Targets
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Summary
• CRISPR system can be heterologously reconstituted in mammalian cells to facilitate efficient genome editing.
• Use RNA to program sequences pecific DNA cleavage defines a new class of genome engineering tools.
• Multiplex genome editing in mammalian cells enables powerful applications .
• Efficiency and versatility could be further improved.
• Potential constraints posed by PAM, crRNA secondary structure or genomic accessibility resulting from chromatin and DNA methylation states.
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Engineer Type II CRISPR System in Human CellsCodon-optimization GFP reporter assay
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Test The Functionality of CRISPR System by GFP Reporter Assay
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Components Required for Genome Editing
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Effects of Cas9D10A Mutant Nickase on NHEJ
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CRISPR MediatedGenome Editing is Sequence-specific
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DNMT3a and DNMT3b Genes Targeting in Human Cells
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Target theAAVS1 Locus in 293Ts, K562 cells, and PGP1 Human (iPS) Cells
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HR mediated integration
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Multiplex Synthesis, Retrieval and U6 Expression Vector Cloning of Guide RNAs Targeting Genes in
The Human Genome
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Summary
• CRISPR-mediated gene targeting for RNAguided, robust, and multiplexable mammalian genome engineering.
• Expand the range of CRISPR-targetable sequences through the use of homologs with different PAM requirements or by directed evolution.
• Inactivating one of the Cas9 nuclease domains increases the ratio of HR to NHEJ and may reduce toxicity.
• Target locus’s underlying chromatin structure and epigenetic state will also affect the efficiency of genome editing in eukaryotic cells.
• Evaluating Cas9 homologs identified through bioinformatics and directed evolution of these nucleases toward higher specificity.
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Parallels and distinctions between CRISPR RNA-guided silencing systems and RNAi
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Steps leading to new spacer integration
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Diverse mechanisms of CRISPR RNA biogenesis
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