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