The CRISPR/Cas9 gene editing tool is a rapid, easy, and efficient way of altering genes of interest in cell lines. Cancer cells develop mechanisms to evade immune attack, and in this project our target genes were the ones responsible for producing the proteins that aid this evasion. A couple of those were CD47 (a protein that blocks macrophages from eating up cancer cells), PD-L1 (a protein that inactivates T cells).

For this project, I developed in-house protocols for efficient CRISPR knockout and analyses. Following are some details:

Cell lines: B16F10 (mouse melanoma), MC38 (mouse colorectal carcinoma), TRAMP-C2 (mouse prostate cancer), EpH4-1424 (mouse mammary carcinoma), B16F10-GMCSF (GVAX)

Guide RNA design: DeskGen, Benchling, Synthego

CRISPR component format:

• All-inclusive plasmid (PX458-Addgene) where the guide RNA had to be inserted using restriction digestion, cloned into competent E. coli, and sequenced.
• Ribonucleoprotein (RNP) complex, where the guide RNA and SpCas9 protein were complexed and delivered directly into cells.

CRISPR cargo delivery:

• Chemical transfection/lipofection using Lipofectamine and Viafect
• Electroporation/nucleofection using the Neon Electroporation system

Analyses:

• DNA – PCR (primers designed using IDTPrimerQuest), T7E1 mismatch assay to calculate indel %, Sanger’s sequencing
• Protein – immunofluorescence using direct and indirect antibody staining and quantification using flow cytometry, analyses by FlowJo

Downstream processes: single cell sorting using FACS, clonal expansion and analysis

Outcomes: An average 85% knockout of cell surface proteins, with knockouts ranging from 55% in TRAMP-C2 cell lines to 96% in MC38 cell lines.