Takara Guide-it CRISPR Genome-Wide sgRNA Library System \5 Screens

The Guide-it CRISPR Genome-Wide sgRNA Library System includes all of the components necessary to perform five lentiviral-based, genome-wide CRISPR/Cas9 knockout screens in human cells. Provided with the kit are lentiviral vectors for expression of Cas9 and >76,000 sgRNAs targeting >19,000 human genes (four guides per gene) in lyophilized, ready-to-transfect formats that simplify the production of high-titer lentivirus. The sgRNA library included with this kit is based on the Brunello library (Doench et Al. 2016), and incorporates design features intended to maximize on-target efficacy and minimize off-target effects. To ensure balanced guide representation, we analyze the sgRNA library by NGS to confirm that >90% of the included sgRNAs fall within a 10-fold distribution range. Enrichment or depletion of sgRNAs in screened cells can be analyzed by NGS with the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647).

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.

Identification and analysis of sgRNAs isolated from cells after a 6-thioguanine (6-TG) screen. Panel A.sgRNA representation was compared between the original plasmid library population used to make the lentivirus preparation and the resulting transduced, selected and non-selected cell populations. All four sgRNAs forHPRT(red dots) andNUDT5(black dots) were enriched in the 6-TG-selected population (blue dots). The representation of these sgRNAs in the plasmid library (gray) and transduced, non-selected population (orange) is also shown.Panel B.After selection, the sgRNA representation was shifted in response (blue) to 6-TG. Individual sgRNAs that were enriched are highlighted within this correlation (red and orange dots). Inset shows fold-enrichment of the four sgRNAs for theHPRTgene as compared to the non-selected population.

Determination of transduction efficiency of the sgRNA library lentivirus using mCherry fluorescence.Lentivirus containing the sgRNA library was produced following the instructions in the user manual. Lentivirus was harvested after 48 hrs and used to transduce Cas9-expressing A375 cells at varying MOIs. Transduced cells were plated and analyzed for transduction efficiency by fluorescence microscopy and FACS after 48 hours.

Vector and sgRNA scaffold design used in the Guide-it CRISPR sgRNA library. Panel A.The pLVXS-EF1a-Cas9-PGK-Puro and pLVXS-sgRNA-mCherry-hyg vectors maps showing the lentiviral vector backbone. The vectors are self-inactivating for increased safety during production and use. The pLVXS-sgRNA-mCherry-hyg vector contains both mCherry and hygromycin markers expressed from an IRES-linked bicistronic expression cassette. The sgRNAs are expressed from a human U6 promoter and contain an optimized scaffold sequence for better Cas9 loading and editing efficiency (Panel B).