Mar 19, 2025

Public workspaceCRISPR-Cas9 Screening Protocol for Gene Perturbation Analysis

Nature Cell Biology
DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmyo9nv3p/v1
  • mai.nguyen 1,
  • heidi.mcbride 1,
  • Sidong Huang2
  • 1Department of Neurology and Neuroscience, Montréal Neurological Institute, McGill University, Montréal, Canada;
  • 2Department of Biochemistry, McGill University, Montréal, Canada
Lilia Rodriguez
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmyo9nv3p/v1
External link: https://doi.org/10.1038/s41556-025-01774-y
Protocol Citation: mai.nguyen , heidi.mcbride , Sidong Huang 2025. CRISPR-Cas9 Screening Protocol for Gene Perturbation Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmyo9nv3p/v1
Manuscript citation:
Mai Nguyen, Jack J. Collier, Olesia Ignatenko, Genevieve Morin, Vanessa Goyon, Alexandre Janer, Camila Tiefensee Ribeiro, Austen J. Milnerwood, Sidong Huang, Michel Desjardins, Heidi M. McBride (2025) MAPL regulates gasdermin-mediated release of mtDNA from lysosomes to drive pyroptotic cell death.Nature Cell Biology doi: 10.1038/s41556-025-01774-y
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 18, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124612
Keywords: ASAPCRN, cas9 screening protocol for gene perturbation analysis, cas9 screening for gene perturbation analysis, cas9 screening protocol, cas9 screening, gene perturbation analysis, crispr, screening
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
This protocol describes CRISPR-Cas9 Screening for gene perturbation analysis.
Materials
  • 2 mg/ml puromycin
  • High Pure PCR Template Preparation Kit (Roche # 11796828001)
  • Phusion HF DNA polymerase (ThermoFisher Scientific #F-530XL)
  • Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher Scientific #Q33232)

ReagentHigh Pure PCR Template Preparation KitRocheCatalog #11796828001

ReagentPhusion™ High-Fidelity DNA Polymerase (2 U/µL)Thermo FisherCatalog #F-530XL

ReagentQuant-iT™ 1X dsDNA HS AssayThermo FisherCatalog #Q33232

Troubleshooting
Procedure
1w 5d 0h 5m
Plate 10 x 150 mm of cells at 8 x 106 cells per dish in complete growing medium.
Incubate DurationOvernight .

Incubation
Overnight
  • Next day, infect cells with human CRISPR Brunello lentiviral pooled library.

Note
Four unique sgRNAs (single guide RNAs) per gene, comprising a total of ~76,000 sgRNAs.

  • Incubate DurationOvernight .
Citation
Doench, J., Fusi, N., Sullender, M. et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology.
https://doi.org/10.1038/nbt.3437
LINK

Incubation
Overnight
The following day, trypsinize cells from all 10 plates, pool and plate into 20 x 150 mm dishes (5 x 105 cells per dish) with growing medium containing Amount2 µL puromycin. Incubate for Duration72:00:00 .

3d
Incubation
Change to regular medium and incubate for Duration168:00:00 to allow complete editing.

1w
Trypsinize all 20 plates, pool and determine cell number.
Plate into 100 x 150 mm dishes (5 x 105 cells per dish).
Infect 50 plates with Ad-rtTA and 50 plates with Ad-MAPL-flag adenovirus at 10 pfu/cell. Incubate for Duration48:00:00 .

2d
Incubation
Change to regular medium and refresh every 2-3 days.

Note
For Ad-rtTA, cells reached confluency in 1.5 weeks and for Ad-MAPL-flag, ~3 weeks.

Harvest cells by trypsinization, pool together all 50 plates, disperse to single cell, and determine cell number.
Make aliquots of 20 x 106 cells and centrifuge for Centrifigation1000 x g, Room temperature, 00:05:00 .

5m
Centrifigation
Remove supernatant and store pellets at Temperature-80 °C .

Temperature
Isolate genomic DNA using High Pure PCR Template Preparation Kit (Roche #11796828001) as described by the manufacturer.
Amplify sequences of the gRNA from genomic DNA by PCR using Phusion HF DNA polymerase (ThermoFisher Scientific #F-530XL) using a 2-step amplification adding a unique 6-bp index per sample and sequencing adapter sequences as described in S. Huang et al. (2012) Cell 151, 937-950.
Quantify using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher Scientific #Q33232).
Sequence amplified gDNA using HiSeq2500 System (Illumina).
Map sequence reads to the library using xcalibr and analyze counts with MAGeCK (version 0.5.8) as described in W. Li et al., (2014) Genome Biol 15, 554.
Analyze
Use Robust Rank Aggregation (RRA) algorithm to identify genes whose perturbation (knockout or overexpression) primarily enhanced fitness in the MAPL overexpressing group but not the control group.
Protocol references
Doench, J.G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature biotechnology 34, 184-191 (2016).

S. Huang et al. (2012) Cell 151, 937-950.

W. Li et al., (2014) Genome Biol 15, 554.