adam clore, phd manager, synthetic biology design flavors.pdf · flavors of crispr. adam clore,...
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Flavors of CRISPR
Adam Clore, PhDManager, Synthetic Biology Design
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Traditional CRISPR Mediated Gene Disruption • Rapid gene disruption in CHO cells• In Zebrafish• In Yeast• Then list goes on…
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CRISPR Mediated Insertions
Non Homologus End Joining (NHEJ)Error prone
Leads to indels and rearrangements
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Dual CRISPER Nickases• D10A mutant of Cas9 target to two
locations that flank the desired site• Optimization of dual nickase
separation distance • Reduction of off target activity 50 -
1500 fold
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CRISPRiCRISPR Based Gene Silencing
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Method• CRISPRi uses a nuclease dead Cas9 Protein to sterically block transcription elongation or
promoter binding• First demonstrated in E. coli by Qi et al (Weissman lab, Berkely) in 2013
http://dx.doi.org/10.1016/j.cell.2013.02.022
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Transcription repression
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Benefits of CRISPRi• Functions in all domains of life
• siRNA is not active in Prokaryotes and some fungi• No permanent change to genome • Different activity than RNAi• Anecdotal information suggests that CRISPR may be more robust that RNAi
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CRISPR activation: CAS9 as a Homing device
• Multiple, tuned, gene activation with nuclease-dead CAS9/gene promoter fusion proteins
• Promoter gene and sgRNA were gBlocks fragments
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CRISPR Visualization: Experimental optimization
• Cas9 – GFP Fusion allows for real time determination of transfection efficiencies and allows for FACs sorting
• Mammalian expression vectors available from Addgene
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CRISPR Visualization: in vivo and Real time • Chen et al. demonstrate real time
resolution of genomic structures in living cells
• Resolve structure of highly repetitive elements (telomers)
• Observe the movement of a single gene throughout mitosis
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Screening with CRISPR Libraries• First large screen done by Zhang Lab in 2014• Created Genome scale CRISPR knockout library (GeCKO)
• 18080 gRNAs targeting all 5’ exons of all genes in the human genome• 3-4 gRNAs per gene
3 JANUARY 2014 VOL 343 SCIENCE
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Library construction and testing• Lentiviral vector with CRISPR cassette• Library construction and screening
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Data• When CRISPR knockouts create lethal deletions the corresponding gRNA are not
observed in the population• Identification of genes associated with vemurafenib resistance
• a therapeutic RAF inhibitor used to treat melanoma • Relative ratios of gRNAs in vemurafenib treated cells vs control indicates genes
conferring sensitivity to the drug
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CRISPER as a Biological Sensor• Paris- Bettencort iGEM team
• 1st place in 2013 Overgrad competition
• Detection of TB drug resistance genes using a Phage-delivered cassette containing
• a Cas9/gRNA targeting a drug resistance gene• a LacZ gene driven by an SOS dependent promoter
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CRISPR as a biological Sensor
• Designed for a quick and inexpensive field diagnostic