Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Abstract

Genome-wide CRISPR phenotypic screens are clarifying many fundamental biological phenomena. While pooled screens can be used to study selectable features, arrayed CRISPR libraries extend the screening territory to cell-nonautonomous, biochemical and morphological phenotypes. Using a novel high-fidelity liquid-phase plasmid cloning technology, we generated two human genome-wide arrayed libraries termed T.spiezzo (gene ablation, 19,936 plasmids) and T.gonfio (gene activation and epigenetic silencing, 22,442 plasmids). Each plasmid encodes four non-overlapping single-guide RNAs (sgRNAs), each driven by a unique housekeeping promoter, as well as lentiviral and transposable vector sequences. The sgRNAs were designed to tolerate most DNA polymorphisms identified in 10,000 human genomes, thereby maximizing their versatility. Sequencing confirmed that ~90% of each plasmid population contained ≥3 intact sgRNAs. Deletion, activation and epigenetic silencing experiments showed efficacy of 75-99%, up to 10,000x and 76-92%, respectively; lentiviral titers were ~10^7/ml. As a proof of concept, we investigated the effect of individual activation of each human transcription factor (n=1,634) on the expression of the cellular prion protein PrPC. We identified 24 upregulators and 12 downregulators of PrPC expression. Hence, the T.spiezzo and T.gonfio libraries represent a powerful resource for the individual perturbation of human protein-coding genes.

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

“…The dual-sgRNA strategy may provide similar benefits for other CRISPR modalities such as CRISPR-mediated overexpression (CRISPR activation [CRISPRa]), as also described by others ( Yin et al, 2022 ), and in mouse cells. We have cloned a human dual-sgRNA CRISPRa library ( Supplementary file 9 ) and designed in silico dual-sgRNA CRISPRi and CRISPRa libraries targeting the mouse genome ( Supplementary files 10 and 11 ).…”

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

“…Examples include screens in primary or stem-cell derived models or in vivo as well as screens with high-content readout such as Perturb-seq (Bock et al, 2022;Przybyla and Gilbert, 2021). Additionally, this dual-sgRNA strategy may provide similar benefits for other CRISPR modalities such as CRISPR-mediated overexpression (CRISPR activation, CRISPRa), as also described by others (Yin et al, 2022), and we have designed a dual-sgRNA CRISPRa library for this purpose (Table S8). Finally, the improved knockdown afforded by the dual-sgRNA approach will also be beneficial in arrayed experiments, in which recombination is not a concern, and we have included a protocol for cloning dual-sgRNA libraries in array (Supplementary Note 3).…”

Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes

“…The identification of known mesenchymal GBM drivers such as WWTR1 31 and FOSL1 32 by MGT#1 and MGT#4, respectively, denotes the specificity of the sLCRs and the need for improving the signal-to-noise ratio in phenotypic pooled screens. The constant development of powerful dCas9 effectors and of arrayed gRNA libraries is likely to improve the discovery power of genome-wide phenotypic screens 42 . Arrayed screens are also anticipated to reduce the influence of non-autonomous phenotypic changes in pooled screens, which are responsible for neutral gRNA background recovery in multicellular 3D models like the one shown here.…”