Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene

CRISPR-Cas9 mediated gene editing in induced pluripotent stem cells became an efficient tool to investigate biological mechanisms underlying genetic-driven diseases while accounting for the respective genetic background. This technique relies on the targeting of a specific nucleotide sequence present in the gene of interest. Therefore, the gene editing of some genes can be complicated by non-coding pseudogenes presenting a high homology of sequence with their respective genes. Among them, GBA is raising special interest because of its implication as the most common genetic risk factor for Parkinson’s disease. In this study, we present an easy-to-use CRISPR-Cas9 gene editing strategy allowing for specific editing of point mutations in a gene without genetic alteration of its pseudogene exemplified by the correction or insertion of the common N370S mutation in GBA. A quality control strategy by combined fluorescence and PCR-based screening allows the early identification of correctly edited clones with unambiguous identification of the status of its pseudogene, GBAP1. Successful gene editing was confirmed by functional validation. Our work presents the first CRISPR-Cas9 based editing of a point mutation in GBA and paves the way for technically demanding gene engineering due to the presence of pseudogenes.


DNA extraction
For DNA extraction, iPSC were harvested with Accutase (Sigma-Aldrich) and pelleted by centrifugation at 300g for 3 minutes. The Dneasy Blood and Tissue kit (Qiagen) was used according to the recommendations. Quantity and purity of DNA was assessed using the ratios 260/280nm and 260/230nm on a Spectrophotometer NanoDrop© 2000C (Thermo Scientific).

PCR Amplification for construction of donor vector:
Purified genomic or plasmid DNA was amplified using the GXL polymerase (Takara) as recommended by the manufacturer. 10ng of purified DNA was used for amplification.

TOPO Cloning:
PCR products were PolyA tailed to allow subsequent insertion in TOPO vector. Subsequently, the PCR products were ligated into pCR2.1-TOPO vector using the TOPO TA Cloning kit (Thermo Fisher Scientific). Briefly, 4µl of polyA-tailed PCR product were mixed with 1µl of TOPO vector and 1µl of salt solution and the reaction was incubated for 30 min at RT allowing the ligation of the PCR product into the TOPO vector via recognition with the thymidine overhangs of the linearized vector.

Bacterial transformation and plasmid DNA extraction
One Shot® TOP10 Chemically competent E. coli (Thermo Fisher Scientific) were transformed using plasmid DNA and kept under ampicillin treatment (100µg/ml). Plasmid DNA from selected colonies was extracted using QIAprep Spin Miniprep Kit (Qiagen) and sequencing was performed at Eurofins Genomics. For preparation of the plasmids for transfection, the selected bacterial clones were allowed to grow in 250 ml of LB Broth with ampicillin overnight and plasmid DNA was extracted using HiSpeed Plasmid Maxi Kit (Qiagen).

Generation of LHA and RHA
The LHA was generated in one step (Fig. S1). The TOPO_HDR was amplified with the primer pair LHA_F/LHA_R with the GXL polymerase (Fig. S1A). The resulting PCR product was polyA tailed and cloned into a TOPO vector. After transformation, 5 independent colonies were screened via amplification with the M13 primers (Fig. S1B). Only the clones presenting a unique band at the expected size of 833bp were submitted to sequencing (LHA + sequence of TOPO between M13 primer binding sites = 663+170bp) with the M13 forward primer (Fig. S1C).
The RHA was generated in two steps (Fig. S1). The TOPO_HDR was amplified with the primer pair RHA_F/RHA_R with the GXL polymerase (Fig. S1A). The resulting PCR product was polyA tailed and cloned into a TOPO vector. After transformation, independent colonies were screened via amplification with the M13 primers (Fig. S1B). Only the clones presenting a unique band at the expected size of 1088bp were submitted to sequencing (LHA + sequence of TOPO between M13 primer binding sites = 918+170bp) with the M13 forward primer. This first step also allowed to silent-mutate the PAM sequence TGG>TCG (Fig. S1D, light blue). In the second step, we generated the TTAA motif and created a RHA to correct the mutation (harbouring AAC codon) or to insert the mutation (harbouring AGC codon) via amplification of the selected TOPO_RHA (Clone_10) with different forward primers (RHAF_step2_N370 and RHAF_step2_N370S) (Fig. S1E). Subcloning into TOPO vector and sequencing with M13 primers allowed us to identify clones with correct sequence for correction of the mutation (Clone_4 = TOPO_RHA_N370) and for insertion of the mutation (Clone_11 = TOPO_RHA_N370S) ( Fig. S1F and G).

Cell culture
The iPSC lines were maintained on Geltrex (Gibco) coated plates under E8 medium composed of DMEM/F12 (ThermoFisher) supplemented with 1% Insulin-Transferrin (Life/Tech), 1% Penicillin-Streptomycin (Life/Tech), 64µg/ml of ascorbic acid (Sigma-Aldrich), 20ng/ml of FGF-2 (Peprotech), 2ng/ml of TGF-β1 (Peprotech) and 100ng/ml Heparin (Sigma-Aldrich). To improve maintenance conditions, 10% of mTesR1 (Stem cell technologies) was added to the abovementioned media. Media was changed every day. Geltrex coating was performed by diluting concentrated matrix in ice-cold DMEM/F12 medium following manufacturer's instructions. The solution was allowed to coat for at least 1h at 37°C. Every 5 to 7 days iPSCs were split at a 1:5 to 1:10 ratio using 0.5 mM EDTA (Sigma-Aldrich). When iPSC were needed as single cells, culture were detached with Accutase (Sigma-Aldrich) and replated in media containing 10µM of ROCK inhibitor Y-27632 (Abcam). Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2.

Transfection of iPSCs
For transfection, 5x10 6 living iPSC were collected with Accutase. Five transfection were realized with 1.10 6 cells per transfection. The Human Stem Cell Nucleofector Kit 1 (Lonza) was used and cells were electroporated using the B16 program from Amaxa nucleofector II (Lonza). After electroporation, cells were allowed to stay in the cuvette at 37°C for 5 min as we observed a better survival after completing this step. Then all eletroporated cells were platted in two Geltrex precoated 1 well plate NuncTM OmniTrayTM (Thermo Fisher Scientific) in media supplemented with ROCK inhibitor.