Mar 19, 2025
CRISPR-Cas9 Screening Protocol for Gene Perturbation Analysis
- mai.nguyen1,
- heidi.mcbride1,
- Sidong Huang2
- 1Department of Neurology and Neuroscience, Montréal Neurological Institute, McGill University, Montréal, Canada;
- 2Department of Biochemistry, McGill University, Montréal, Canada
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
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 17, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124612
Keywords: ASAPCRN
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)
High Pure PCR Template Preparation KitRocheCatalog #11796828001
Phusion™ High-Fidelity DNA Polymerase (2 U/µL)Thermo FisherCatalog #F-530XL
Quant-iT™ 1X dsDNA HS AssayThermo FisherCatalog #Q33232
Procedure
Procedure
1w 5d 0h 5m
1w 5d 0h 5m
Plate 10 x 150 mm of cells at 8 x 106 cells per dish in complete growing medium.
Incubate 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 Overnight .
CITATION
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 2 µL puromycin. Incubate for 72:00:00 .
3d
Change to regular medium and incubate for 168:00:00 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 48:00:00 .
2d
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 1000 x g, Room temperature, 00:05:00 .
5m
Remove supernatant and store pellets at -80 °C .
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.
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.