CASE STUDY: CRISPR/Cas9 Cell Line Model Generation (Gene Knock-in/ Insertion)

Large Fragment Knock-in Cell Line Model Using CRISPR/Cas9

Goal: To insert a large fusion gene fragment (8.5 kb) downstream of a specific endogenous promoter in a cancer cell line using CRISPR/Cas9.

Briefly, gRNAs were designed for the targeted knock-in site and validated using next generation sequencing (NGS). The gRNA with the highest NHEJ frequency (normalized to control transfection) was used for transfection along with corresponding donor plasmid and Cas9 into the chosen cell line. After electroporation and single cell cloning, three positive heterozygous clones were identified and confirmed for gene insertion using PCR and Sanger Sequencing (data not shown).

Figure A. Schematic Representation of the 8.5 kb knock-in fragment and genotyping scheme.

After transfection, the pooled cells were analyzed for insertion of the gene of interest using PCR using primers for left (LA) and right (RA) arms. The wild type (WT) cells did not show PCR product. The transfected cells (+) showed the PCR fragment sizes: ~1.1 kb for LA and ~ 1.5 kb for RA indicating a positive insertion of transgene. These cells were then subjected to single cell cloning to identify positive single clones.

Figure B. PCR gel electrophoresis of wild type (WT) cells and transfected cells containing gene knock-in (+).