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Mitochondrial and ER membrane protein trafficking CRISPR screens

Citation

Yao, David; Coukos, Robert (2022), Mitochondrial and ER membrane protein trafficking CRISPR screens, Dryad, Dataset, https://doi.org/10.5061/dryad.tb2rbp00n

Abstract

The trafficking of specific protein cohorts to the correct subcellular location at the correct time is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or FACS. To broadly empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identify genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in the mislocalization and destabilization of mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

Methods

This dataset contains FASTQ files from several HiLITR CRISPR screens. K562 cell lines stably expressing various HiLITR configurations were transduced with lentiviral CRISPRi sgRNA libraries (either genome-wide, and/or "batch" libraries) at a low multiplicity of infection such that each cell received one sgRNA on average. After 48 hours, cells with successful lentiviral sgRNA integration events were selected for using Puromycin over the next 96 hours. In general, cells were induced with doxycycyline to induce expression of the HiLITR TEV protease 16-24 hours prior to light stimulation (some differences in light induction timing exist depending on the cell line). After an incubation period of 8-16 hours to allow HiLITR reporter expression to occur, cells were sorted by FACS into bins based on levels of HiLITR reporter expression. The genomic DNA was extracted from the sorted populations, and the integrated sgRNAs in each population were amplified, barcoded, and sequenced on an Illumina NextSeq instrument at various read depths (but always greater than or equal to 300X sequencing coverage for each sgRNA in the lentiviral sgRNA library transduced into a given cell line). Experiments were performed in technical replicates. The sequencing .bcl files were converted to FASTQ files using the Illumina bcl2fastq conversion software

Usage Notes

C25 FASTQ files belong to the ER batch CRISPR screen. M2C9 FASTQ files belong to the TA screen. M13C2 FASTQ files belong to the SA screen. TR refers to technical replicates, of which there are 2 for each experiment.