Jun 16, 2026
Generation of the HAP1 ATG9A-GFP cell line
- Daniel Bernklau1,
- Julia Romanov1,
- Elisabeth Holzer1
- 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Protocol Citation: Daniel Bernklau, Julia Romanov, Elisabeth Holzer 2026. Generation of the HAP1 ATG9A-GFP cell line. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46j4ogo5/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
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Created: August 08, 2025
Last Modified: June 16, 2026
Protocol Integer ID: 224541
Keywords: ASAPCRN, generation of the hap1 atg9a, gfp cell line this protocol, gfp cell line, hap1 atg9a, cell, description of generation, protocol detail
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol details the description of generation of the HAP1 ATG9A-GFP cell line.
Materials
- Culture dishes (Eppendorf #0030702115)
- FuGENE 6 transfection reagent (Promega, Cat# E2691)
FuGENE(R) 6 Transfection Reagent, 1mlPromegaCatalog #E2691
- OptiMEM (Gibco 11058021)
Opti-MEM™ I Reduced Serum Medium, no phenol redThermo FisherCatalog #11058021
- Proteinase K (2mg/ml in MiliQ, Sigma-Aldrich P6556)
Proteinase KMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6556-100mg
- gDNA isolation (Thermo Scientific Gene Jet #K0721)
GeneJET Genomic DNA Purification KitThermo FisherCatalog #K0721
Construction of the homology repair template
Note
The plasmid based repair template consists of the following blocks.
A pUC-19 vector serves as backbone for the homology template.
Genomic dna (~1400bp) upstream of the ATG9A Stop codon, the CDS of monomeric EGFP, a 3C cleavage site, a FLAG tag and genomic DNA (~1500bp) downstream of and including the ATG9 stop codon. Introduce linkers between the relevant blocks (RRID: Addgene: 244942). Mutate PAM sites (silent if in exon; here, one site was mutated).
To generate a nick at the desired locus select two guide RNAs close to the region of ATG9A Stop codon by CRISPick and clone into AIO-Puro (RRID: Addgene: 74119) according to the instructions.
Transfection of the CRISPR plasmids
45m
Grow Hap1 wt cells in 10 cm culture dishes (Eppendorf #0030702115) until ~70% confluent.
Change the medium to fresh IMDM +P/S +10%FCS 00:30:00 before the transfection.
30m
Add 30 µL of FuGENE 6 transfection reagent (Promega, Cat# E2691) to 500 µL OptiMEM (Gibco 11058021).
Add 5 µg of AIO-Puro containing gRNAs and 5 µg of homology template plasmid to 500 µL of OptiMEM.
Incubate for 5' individually at Room temperature .
Mix the solutions and incubate for 00:15:00 at Room temperature .
15m
Add dropwise to the cells.
Additionally prepare non-transfected control cells.
Selection of transfected cells and enrichment of GFP-positive cells
3d
24:00:00 after transfection change the medium to IMDM +P/S + 10%FCS containing 5 µL Puromycin.
1d
Apply selection for 48:00:00 or until non-transfected control cells are dead.
2d
Enrich for GFP positive cells via FACS bulk sort.
Expand cells in a 10 cm dish until confluent.
Selection of CRISPR-tagged cell clones
1h 15m
Subject cells to FACS single cell sort into 96w plates.
Expand growing clones and test for integration of the homology arms by PCR.
Prepare crude gDNA containing samples for this PCR as follows (from 96-well plate).
Remove the medium and wash 1x with DPBS.
Add 20 µL Trypsin and incubate at 37 °C for ~3'.
Stop the Trypsin with 100 µL of medium and pipette thoroughly.
Transfer the suspension to a PCR plate and cover with a foil.
Spin the plate for 2' in a plate centrifuge.
Remove the medium carefully and wash with PBS.
Remove the medium carefully and wash with PBS (1).
Remove the medium carefully and wash with PBS (2).
Add 25 µL of MiliQ.
Boil at 95 °C for 00:05:00 .
5m
Cool down On ice and add 5 µL of proteinase K (2mg/ml in MiliQ, Sigma-Aldrich P6556).
Incubate at 55 °C for 01:00:00 .
1h
Stop the enzyme reaction at 95 °C for 00:10:00 .
Note
This is a very crude gDNA sample and serves for a quick initial check. Use 2.5 µL gDNA prep as template for 12 µL of PCR volume.
10m
Then test for the correct locus integration by PCR.
Assess the protein size and levels of ATG9-GFP as compared to WT cells.
Subject the clones passing this quality control step to a starvation assay checking the levels of autophagy markers.
Also test cells for undesired Cas9 integration into the genome by WB.
Subject the positive clones (correct integration, expression of tagged protein at endogenous levels, typical protein pattern in starvation assay, negative for Cas9) to further validation by gDNA isolation (Thermo Scientific Gene Jet #K0721), PCR amplification of the locus and expanded sequencing of the integration site.