gRNA library design and construction

A genome-wide library was designed with five gRNAs against each gene using the Illumina Human BodyMap 2.0 and NCBI CCDS data sets. The genome-wide library contains ∼90,000 gRNAs elements covering 18,360 genes. The library was synthesized using chip-based array oligonucleotide synthesis to generate spacer-tracrRNA-encoding fragments⁷⁶. gRNAs were cloned in pool format between BpiI sites of the pNGx-LV-g003 vector derived from pRSI16 lentiviral plasmid by Cellecta Inc. gRNA sequences are driven by the U6 promoter. The UbiC promoter drives the expression of RFP fused via T2A to a Puromycin resistance cassette. Cells transduced with gRNA expressed RFP and can be selected using Puromycin antibiotic. The custom array mini pool library contains 5180 gRNA elements, with ten gRNAs targeting each gene. These were cloned as described for the genome-wide library for the generation of spacer-encoding fragments that were PCR-amplified and then cloned into the BbsI site of pNGx-LV-g003 lentiviral plasmid²³. gRNA sequences were design to target the most proximal 5′ exons of individual genes. Sequencing of genome-wide library plasmids revealed a normal distribution and passed all quality checks before being packaged into lentivirus.