Speaker: Jiang-An Yin，Ph.D.， Senior Research Scientist， University of Zurich

Time: 10:00-11:30 a.m., July 18, 2024, GMT+8

Venue: Room B117, Research Complex #2, PKU

Abstract: 

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable phenotypes. However, arrayed CRISPR libraries are very limited, and the generation of such libraries requires assembling thousands of sgRNA-expressing vectors. Using a newly invented massively parallel plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). Each plasmid encodes an array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92%, and ~10,000-fold in deletion, silencing, and activation experiments, respectively. An arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the prion protein PrC. Furthermore, screening with a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two existing libraries. Finally, we show that "postpooling" individually produced lentiviruses eliminates template-switching artifacts and enhances the performance of pooled CRISPRoff screens. These versatile libraries represent a powerful resource for the targeted perturbation of human protein-coding genes.

Source: Biomedical Pioneering Innovation Center, PKU