Figure 4.

Human CRISPR library construction and sgRNA read distribution. (A) Schematics of sgRNA oligo design. The pre-designed sgRNA 20-mers are flanked by 5΄- and 3΄-PCR primers and BsmBI sites. (B) Schematics of parallel retrieval of oligo pools. Library oligos with different flanking primer sequences were synthesized together as a single 12k oligo pool. Specific primer pairs were used to PCR amplify and retrieve subsets of oligos from the master pool. The PCR amplicons were cloned into lentiviral vectors using Golden Gate assembly. (C) Distribution of normalized sgRNA read frequency from 13 distinct plasmid sub-pools generated from a single oligo pool. (D) Percentage of sgRNA reads that are within an 8-fold range from 24 plasmid sub-pools generated from two master oligo pools.