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GENERATION OF KNOCKOUT CELL LINES FOR INVASION-REGULATING SCAFFOLD GENE AFAP1L1 INSARCOMA CELLS BY CRISPR/CAS9 TECHNOLOGY
Rebecca ShapiroKalamazoo CollegeEvan Ingley, Ph.DHarry Perkins Institute of Medical ResearchPerth, Western Australia
Introduction• Sarcoma and U2OS cell line• AFAP1L1 and metastasis
CRISPR/Cas9• CRISPR/Cas9
• 1987 in Ishino lab• CRISPR locus• Endonuclease (Cas9)• DSB can be repaired by
non-homologous end joining (NHEJ) or homology directed repair (HDR)
Present Study Overview• Homology Directed
Repair (HDR) Pathway used
• mCherry plasmid insert• Fluorescence Activated
Cell Sorting (FACS) • PCR• Sequencing verification
https://www.addgene.org/CRISPR/guide/
Designing CRISPR plasmid
Sigma Aldrich, 2015.
Continued Methods Overview
• Transformation and colony screening• Plasmid preparation• Sequencing for confirmation• Co-transfection into U2OS cells• FACS Sorting
5’ CACC GTACCTCAGCGATACCACCC 3’ Top5’ CATGGAGTCGCTATGGTGGG CAAA 3’ Bottom
Co-transfection results Pre-FACS
Cell Culture Growth
Summary• CRISPR plasmid was able to target AFAP1L1 successfully• mCherry repair plasmid confirmed
• 99% positive red fluorescent U2OScells• CRISPR has been proven to be a more effective gene
engineering tool• Future directions
• Sequence verification of each clone• Observing U2OS cell morphology without AFAP1L1• Potential in vivo models (mice) and observe metastatic effect
Thank you!
• SIP Advisor• Dr. M. Wollenberg
• SIP Review Team• Marie Bunker, • Alec Duffey, • Marie Hallinen • Jordan• Henning, • Margaret Rice• Emily Sklar
• Referee Group• Camryn Romph• Louise Silverman• Marie Bunker
• Friends and family!
• Ingley Lab• Evan Ingley, Ph.D• Cindy Le• Matt Lee• Janice Lam, Ph.D
• Harry Perkins Institute of Research
• Hearst Undergraduate Research Fellowship