2.CRISPR theory mechanism and applications (1).pptx - CRISPR-Cas9 The world leader in serving science theory\/mechanism and applications to gene editing

2.CRISPR theory mechanism and applications (1).pptx -...

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The world leader in serving science CRISPR-Cas9: theory/mechanism and applications to gene editing and expression Aaron Chen, Ph.D. Field Application Scientist, Level Biotechnology Inc.
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2 Outline Background of CRISPR/Cas9 Applications of CRISPR/Cas9 Getting started with your experiments Publications of CRISPR/Cas9
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3 Outline Background of CRISPR/Cas9 Applications of CRISPR/Cas9 Getting started with your experiments Publications of CRISPR/Cas9
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4 What is Gene/Genome Editing? • A process whereby researchers can introduce a modification into an endogenous gene • Disruption , Insertion , Replacement at a locus in the genome – Control gene expression – Create SNP – Create Reporter fusions while maintaining endogenous gene regulation
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5 What is CRISPR (CRISPR-Cas; CRISPR-Cas9)? Mechanism of adaptive immunity in bacteria and archaea Evolved to adapt and defend against foreign genetic material (e.g. phage) Several different types of CRISPR pathways in bacteria and archaea Type II: CRISPR-Cas9. Creates a double-strand break in the targeted DNA CRISPR: C lustered R egularly I nterspaced S hort P alindromic R epeats Cas proteins: C RISPR- A ssociated proteins
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6 Why is there a “CRISPR Craze”? Cas9 can be programmed to perform gene editing in “mammalian cells” . Changing a short RNA sequence can easily target to a different site in the genome Simpler and easier than other genome editing technologies (ZFN, TALENs) “unprecedented efficiency and stunning ease of use” ~ Science (2014) 344(6185):707-8 Gene therapy is back! SCIENCE VOL 341 23 AUGUST 2013
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7 How does CRISPR-Cas9 edit genome? Natural bacterial system (Type II): crRNA + tracrRNA + Cas9 protein Two components: single chimeric guide RNA + Cas9 protein Design crRNA to target any sequence next to a PAM (NGG/NCC) in the genome. Cas9 creates a double strand break (DSB) in the genome . DSB occurs on both strands, 3 base pairs upstream of the PAM. DSB is repaired by either NHEJ or HR tracrRNA: trans-activating CRISPR RNA (tracrRNA) PAM: Protospacer Adjacent Motif
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8 Two major repair pathways of DSBs NHEJ HR
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9 Outline Background of CRISPR/Cas9 Applications of CRISPR/Cas9 Getting started with your experiments Publications of CRISPR/Cas9
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10 CRISPR/Cas9 system is a genome editing tool
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11 Applications using CRISPR/Cas9 system 1. Gene disruption (without donor template DNA) 2. Gene knock-out (with a reporter knock-in) 3. Non-protein Coding Gene disruption
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12 Applications using CRISPR/Cas9 system 4. Specific mutations (1) Desired SNP introduction or correction (2) Desired insertions/deletions (3) Tagging the endogenous genes (e.g. HA tag, Flag tag…) 5. Promoter Study - Luciferase replaced the 5’ exon
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13 Applications using CRISPR/Cas9 system 6. Conditional knockout - For essential genes or tissue-specific study inserting LoxP sites around the exon to be knocked-out 7. Large chromosomal deletions - using two sgRNAs to induce DSBs at sites that flank the region of interest Nucl. Acids Res. June 6 (2013)
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