Representation of sgRNAs within the Guide-it CRISPR Genome-Wide sgRNA Library System.
Representation of sgRNAs within the Guide-it CRISPR Genome-Wide sgRNA Library System. Panel A. Guide representation in plasmid library. The Brunello-based sgRNA library was cloned into the pLVXS-sgRNA-mCherry-hyg Vector and amplified. The representation of the sgRNAs within the plasmid DNA library was verified by NGS. The representation of >90% of all the stated sgRNAs within the library was within a 10-fold distribution range. Bars represent the number of sgRNAs with a specific read count. Panel B. Comparison of guide representation in the plasmid library and transduced cells. Genomic DNA was isolated from Cas9+/sgRNA+ A375 cells selected on hygromycin and PCR amplified. The PCR product was sequenced to determine read counts of each integrated sgRNA relative to the starting plasmid DNA population. We observed strong Spearman and Pearson correlations indicating that the system is able to maintain sgRNA representation in the transduced and selected cell population.