K-ras locus in human colon cancer cell line, HCT116, was knocked-out by creating indels in exon 4, as shown in the targeting strategy (Fig. 1).
Viral encapsulation of Cas9 and gRNA delivery particles were carried out, viral titer was optimized for HCT116 cells.
Individual clones were Sanger sequenced to select for homozygous knock-out indels of K-ras at exon 4. Sanger sequencing data (Fig. 2).
Absence of K-ras expression in the selected clone was confirmed via Western Blot (Fig. 3).
Case Study II
A sequence optimized gRNA was designed and synthesized to target a specific region on the GS allele. DG44 cells were transfected with the construct and the cell pool was analyzed by Sanger sequencing.
Several single clones were obtained and Sanger sequence analyzed. A single clone containing a frame shift mutation was carried forward (Fig. 1).
The GS knockout clone was also analyzed for GS protein expression (Fig. 2).
To assess loss of function, GS knockouts were grown in the presence or absence of increasing concentrations of glutamine (Fig. 3). GS knockouts were unable to grow in the absence of glutamine. However growth improved in the presence of increasing concentrations of glutamine, thereby indicating a functional loss in the GS knockout cell line.
In conclusion, successful targeting of GS generated a single clone that was fully validated for lack of expression and GS function.
Case Study III
gRNA pairs are designed to target the flanking locus of a full gene to delete a full gene on genome. gRNAs pairs are optimized with highest cleavage efficiency in the Jurkat cells by GenCRISPR™ technology (Figure A).
The paired gRNAs are co-transfected to the Jurkat cells and enriched by FACS sorting. The deletion efficiency is evaluated by PCR. It is 21% in the transfected cell pool as calculated from light intensity of band (Figure B).
200 single clones are screened with PCR in a high-throughput way and a few of full-allele deletion clones are identified and confirmed by Sanger sequencing and RT-PCR (Figure C).