Minimized combinatorial CRISPR screens identify genetic interactions in autophagy

Abstract

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.

Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3

“…To identify critical autophagy genes for AML cell proliferation we performed a CRISPR/Cas9 knockout screen in the two human AML cell lines THP-1 and MV4-11 (schematized in Figure 1 A). A library targeting 192 autophagy-related genes including core autophagy genes, autophagy receptors, autophagy regulating transcription factors, and ubiquitin-specific proteases [ 22 ] was employed. The library contained four different single guide RNAs (sgRNA) per gene, 876 sgRNAs in total including controls.…”

“…However, this paradoxical role of autophagy depends on the stage of tumorigenesis as it is a pro-survival pathway for established leukemia [ 44 ]. Several CRISPR/Cas9 screens with genetic knockout of various core autophagy genes resulted in a strong autophagy inhibition which negatively correlated with proliferation [ 22 , 45 ]. Similarly, among the 23 identified dropout hits from our screen, we found four core autophagy genes to be essential for AML cell survival ( Figure 1 B,C).…”

FBXL4 deficiency promotes mitophagy by elevating NIX

“…SgRNAs targeting 606 Ubiquitin E3 ligases, included 243 non-targeting control (NTC) sequences were designed by Azimuth2 (Doench et al , 2016) and the top 4 picks (on-target scores>0.6) were chosen per gene, extended by 5 and 3 prime 3Cs homology and obtained as a pool from Twist Bioscience ( Dataset EV1 ). The E3-targeting sgRNA library was made by 3Cs, as described previously (Diehl et al , 2021; Wegner et al , 2019; Wegner et al , 2020). In brief, 3Cs-DNA was generated by mixing phosphorylated pre-annealed oligonucleotides (sgRNA-encoding) with ssDNA (library template plasmid).…”

Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants

“…Existing tools that assist in determining an appropriate sample size for multiplex CRISPR/Cas screens in mammalian cells are not applicable to screens in plants due to different experimental protocols. Additionally, focus on the exploration of the complete combinatorial design space is lacking in these studies (Nagy and Kampmann, 2017 ; Shen J. P. et al, 2017 ; Imkeller et al, 2020 ; Diehl et al, 2021 ).…”