Enabling multiplexed CRISPR/Cas9 screening via massively parallel in-library ligation

Here we developed a massively parallel in-library ligation methodology to simultaneously perturb four pre-designed targets in CRISPR/Cas9 screening. Thousands of pairs of sequences precisely ligated with their counterparts in library, which enabled simultaneous expression of four gRNAs from each single vector. We demonstrated this novel method with 6,236 4-gene combinations targeting 1,599 immune response related genes, and generated a plasmid library with 1,400x coverage. The library performance was evaluated in a canonical T cell activation experiment, and combinations involved in TCR signaling pathway or TCR complex were successfully identified as positive regulators. Novel combination that is reflecting a potential pathway crosstalk was also verified. This new methodology expands the capacity of the perturbation in CRISPR screening and provided a powerful tool for researches in broad fields to study the combinatorial outcomes from coordinated gene behaviors. We examined the library quality in multiple steps over the course of library preparation, including the plasmid library after the 1st golden gate and the final plasmid library after the 4th golden gate. To perform the multiplexed screening, the 4-gRNA expression vectors were transduced to the Jurkat cells with stable Cas9 expression (referred as Jurkat-Cas9 hereafter). After 10-days cell culture and antibiotic selection, cells were stimulated by T Cell Activator. Twenty-four hours after stimulation, the cell activity was assessed based on the surface CD69 expression. Both the top 25% (CD69+) and the bottom 25% (CD69-) cells were collected by cell sorter. NGS sequencing libraries were generated using genomic DNAs collected from the cell populations before-stimulation (Control), CD69+ (Top 25%) post-stimulation and CD69- (Bottom 25%) post-stimulation. The distributions of the 4-gRNA combinations were examined and compared across libraries.The G12 library and G23 library of each sample were treated as technical replicates. For each 4-gene combination, the log2 fold-change of the normalized read counts were calculated between the CD69+ and CD69- samples, and mean of replicates was used. Please note that processed data generated from multiple samples is linked as Series supplementary file and is indicated in the corresponding sample description field.