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