Rapid CRISPR/Cas12a-based dual-crRNA library screening in high fidelity reveals novel genetic interactions during hepatocarcinogenesis [guide-seq]

CRISPR/Cas12a-based combinational screening has shown remarkable potential in identifying genetic interactions. Here, we described an innovative method for combinational genetic screen with rapid constructing of dual-crRNA library by gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which was identified previously by us with strict PAM recognition and low off- targeting, to guarantee the fidelity and efficiency. The custom, pooled SOE crRNA array (SOCA) library for double knockout screen could be conveniently constructed in lab for widespread use and CeCas12a mediated high fidelity screen display good performances even under negative selection screen. By designing an SOCA dual-crRNA library which covered the most of kinase and metabolism-associated gene targets of FDA- approved drugs that were implicated in hepatocellular carcinoma (HCC) tumorigenesis, novel cross talks between the two gene sets were negatively selected out to synergistically inhibit HCC cell growth in vitro and in vivo and also validated by virtual double knockdown screening based on TCGA databases. Thus through our rapid, efficient and high fidelity double knockout screening system, it is very promising to systemically dig genetic interactions between multiple gene sets or synergistic combinations of FDA-approved drugs for clinical translational medicine in the future. The main objectives of this study were (i) to determine whether SOE PCR could successfully construct a double knockout library with complete coverage and (ii) to demonstrate the efficiency and accuracy of SOCA library-based combinational genetic screening. We performed T7E1 assay, targeted deep sequencing and GUIDE-seq to identify the library candidate crRNA with high cleavage activity and Cas12a nucleases with high editing efficiency and low off-target rate and ensured the efficiency and accuracy of screening results. We constructed primer pool by SOE PCR to amplify core fragments (DR-SpacerX-DR-Overlaps-DR- SpacerY-DR), and obtain an entire SOE crRNA array (SOCA) library using infusion cloning. After that, using the SOCA library, we performed in vitro negative screening in the murine HCC cell line (Hepa1-6 cell) and analyzed the correlation between two biological replicates and between two Cas12a nucleases. For in vivo screening, we transplanted target cells into 5-6 nu/nu mice per group and harvested tumors at different time of tumor growth, and then we extracted the genome of the tumor cells and analyzed the crRNA abundance. We performed in vivo and in vitro assays to verify the synergistic interaction of the novel identified combinations and the accuracy of our library screening.