A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Abstract

The ability to map genetic interactions has been essential for determining gene function and defining biological pathways. Therefore, a system to readily perform genome-wide genetic modifier screens in human cells is a powerful platform for dissecting complex processes in mammalian cells, where redundancy and adaptation commonly mask the phenotype of a single genetic perturbation. Here, we report a CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, that can be used to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. As a proof of principle, we apply our approach to study the pathways that mediate tail-anchored (TA) protein insertion at the endoplasmic reticulum (ER). We show that this dual-guide library approach can be successfully coupled with FACS-based reporter screening, to identify genetic epistasis and thereby place TA biogenesis factors in their respective parallel pathways. We demonstrate that this dual-guide approach is both more sensitive and specific than traditional growth screening approaches, and is ideally suited for dissecting the complex interplay between factors in human cells.

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins

“…Consistent with earlier biochemical data, we find that both our forward and arrayed screens show that TA biogenesis depends on the hydrophobicity of their TMDs Rao et al, 2016;Shao et al, 2017;Wang et al, 2010). TAs with sufficiently hydrophobic TMDs (VAMP2) rely on the GET pathway, low hydrophobicity TAs (SQS) rely on the EMC pathway, while intermediate hydrophobicity TAs (Sec61b) can utilize both (Guna, Page, et al, 2023). Unexpectedly, loss of the GET pathway insertase GET1/2 also appeared to have a small effect on biogenesis of several multipass substrates (e.g., the GPCR AGTR2 and EAAT1) in both the genome-wide and arrayed screens.…”

“…In the panel were multipass proteins in which the N-terminus must be translocated across the ER membrane (i.e., Nexo topology: several GPCRs, ORF3a, and M); multipass proteins in which the N-terminus will remain in the cytosol (Ncyt topology: TRAM2, EAAT1, GET2, and YIPF1); and single spanning (Type II: ASGR1; Type I: TRAPα); and TA proteins (SQS, VAMP2, and Sec61b). In order to allow for direct comparison, all reporters contained a full length GFP with the exception of Sec61b, whose targeting is affected by fusion with a florescent protein and therefore required use of the split GFP approach (Figure 1A) (Guna et al, 2022;Guna, Page, et al, 2023;Inglis et al, 2020).…”

“…To delineate which of these associated factors are phenotypically important, we developed a dual-guide CRISPRi approach to systematically identify genetic modifiers of the EMC genome-wide (Figure 3B) (Guna, Page, et al, 2023). Briefly, we generated a library that expresses two sgRNAs on a single plasmid: (1) a genetic anchor guide, targeting the core subunit, EMC2, which when depleted results in loss of the remaining EMC subunits (Pleiner et al, 2021;Volkmar et al, 2019); and (2) a second randomized guide, targeting all open reading frames genome wide using the existing CRISPRi-v2 library (Horlbeck et al, 2016).…”

“…Additionally, we note that in the arrayed screen in Figure 2, all substrates except for SEC61β contain full GFP or RFP fusions. The SEC61β reporter has been previously described (Guna, Page, et al, 2023). Briefly, the TMD and flanking regions were inserted downstream of the first 70 residues of the flexible cytosolic domain of SEC61β.…”

“…For SARS-CoV-2 M and ORF3a, screens were performed with the single CRISPRi-v2 library. We have previously demonstrated that the additional non-targeting guide in the dual guide cassette does not appreciably alter knockdown efficiency of the second guide in the cassette (Guna, Page, et al, 2023). CRISPRi libraries (single CRISPRi-v2 library, Non-targeting dual library [Addgene #197348], or EMC2 dual library [Addgene #197349]) were transduced at a multiplicity of infection less than one into 300-330 million K562-CRISPRi-Tet-ON cells containing the appropriate reporter.…”