Generation of Genetically Tailored Porcine Liver Cancer Cells by CRISPR/Cas9 Editing

Abstract

Pigs provide a valuable large animal model for several diseases due to their similarity with humans in anatomy, physiology, genetics and drug metabolism. We recently generated a porcine model for TP53R167H and KRASG12D driven hepatocellular carcinoma (HCC) by autologous liver implantation. Here we describe a streamlined approach for developing genetically tailored porcine HCC cells by CRISPR/Cas9 gene editing and isolation of homogenous genetically validated cell clones. The combination of CRISPR/Cas9 editing of HCC cells described herein with the orthotopic HCC model enables development of various porcine HCC models, each with a specific mutational profile. This allows modeling the effect of different driver mutation combinations on tumor progression and in vivo testing of novel targeted therapeutic approaches in a clinically relevant large animal model.

Effect of CRISPR Knockout of AXIN1 or ARID1A on Proliferation and Migration of Porcine Hepatocellular Carcinoma

“…A previously validated gRNA was used to target porcine ARID1A gene ( 23 ). Transfection of A272 and A274 porcine HCC cells with Cas9 complexed with ARID1A gRNA resulted in 89% and 99% editing, respectively, at the expected target region as demonstrated by NGS ( Figures 4A, B ).…”

“…Porcine HCC cells were developed from Oncopigs by collagenase digestion of a surgically resected liver section, followed by induction of transgene expression in isolated hepatocytes using Cre recombinase, as previously described ( 17 , 23 ). Porcine HCC cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin.…”

“…gRNAs were designed using CRISPOR ( ) as previously described ( 23 , 24 ) and the AltR ® CRISPR/Cas9 system (IDT Corporation, IL, USA) was used for gene editing. Each gRNA was synthesized by incubating equimolar ratios of crRNA (sequences in Table 1 , synthesized by IDT Corporation) and tracrRNA (#1075927; IDT Corporation) at 95°C for 5 minutes followed by cooling to room temperature.…”

“…Genomic DNA was extracted from porcine HCC cells using QuickExtract DNA Extraction Solution (#QE09050; Lucigen, Middleton, WI) as previously described ( 23 ). The genomic locus that flanks the Cas9 target site was amplified by PCR for 28 cycles using primers listed in Table 1 .…”

Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy

“… [ 79 ] Liver cancer ARID1A, HCC Pig Knockout - CRISPR/Cas9 editing of pig liver cancer cells to create genetically customized cancer cells. [ 80 ] …”