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Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour - PubMed

  • ️Tue Jan 01 2019

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

Jamie R Bhagwan et al. F1000Res. 2019.

Abstract

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca 2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

Keywords: AAVS1 safe harbour; Human induced pluripotent stem cells; CRISPR/Cas9; gene targeting; silencing; stem-cell derived cardiomyocytes; stem-cell derived haematopoietic cells.

Copyright: © 2020 Bhagwan JR et al.

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Conflict of interest statement

No competing interests were disclosed.

Figures

Figure 1.
Figure 1.. Generation of AAVS1-targeted hiPSC clones in an isogenic MYH7 C9123T background and an isogenic ACTC1 G301A background.

( A) Schematic illustrating the chromosomal location of the AAVS1 safe harbour locus. This site was targeted using two sgRNAs in a CRISPR Cas9 nickase strategy. PAM site #1 was silently mutated (G→C) in the targeting construct to prevent it being cut by Cas9 nuclease during targeting. The inserted cassette consists of R-GECO1.0 IRES-Puromycin driven by the CAG promoter. This is flanked on each side by 1 kb of homology to the AAVS1 locus. In ( B) and ( C) confirmatory 5’ and 3’ targeting PCR screen is shown using genomic DNA isolated from the MYH7 C9123T RGECO1.0 isogenic trio (left) and the ACTC1 G301A RGECO duo (right) hiPSCs. Correct 5’ targeting is indicated with a 1221bp product, with sequencing confirming the fidelity of the junction between the AAVS1 left arm homology and the start of the CAG promoter. Correct 3’ targeting is indicated with an 1186bp product, with sequencing confirming the fidelity of the junction between the puromycin-SV40 pA sequence and the AAVS1 right arm homology. ( D, E) Confirmatory PCR and sequencing of hiPSC clones to check 5’ and 3’ targeting of the AAVS1 locus with the HOXA9-T2A-mScarlet cassette.

Figure 2.
Figure 2.. Immunocytochemistry-based screening of AAVS1-targeted clones showing differential expression of R-GECO1.0 between and within cell lines.

( A) AAVS1 targeted hiPSC clones dual-stained for OCT4 (green) and R-GECO (red) to find the highest R-GECO-expressing clone within the five cell line genotypes. High content image analysis identified that ACTC1 WT/WT clone 3 had the highest percentage of pluripotent (94.53% OCT4+) and R-GECO (91.06% ±1.73%) hiPSCs. ACTC1 WT/MUT clone 12 clone had the highest expression of R-GECO (49.37% ±1.33%). Mean ±SD, n = 3 wells. One-way ANOVA with Dunnett’s multiple comparison test, * p ≤ 0.0259; ** p < 0.0045; **** p < 0.0001. Scale bars = 50 µm. ( B) Biallelic targeting PCR screen on isolated gDNA from targeted ACTC1 WT/WT hiPSC clones showing homozygous clones failure to generate the 517bp PCR product. ( C) Biallelic targeting PCR screen showing that all ACTC1 WT/MUT clones tested resulted in a 517bp product and were therefore heterozygous for AAVS1 targeting. L – 1kb ladder; N – no template control; U – untargeted cell line; + - AAVS1 biallelic positive control. ( D) Screening MYH7 MUT/MUT clones using immunocytochemistry on differentiated hiPSC-CMs reveals a significant increase in R-GECO1.0 expression in the MYH7 MUT/MUT Hom 2 clone. Mean ±SD, n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.006; **** p < 0.0001. Scale bars = 50 µm.

Figure 3.
Figure 3.. Changes in transgene expression during differentiation and antibiotic selection.

( A) Immunocytochemistry using an anti-RFP antibody to detect R-GECO1.0 expression shows a reduction in signal in MYH7 WT/MUT 8 and MYH7 MUT/MUT 15 cell lines upon differentiation from hiPSCs to hiPSC-CMs. Mean ±SD, n = 3 technical replicates. One-way ANOVA with Sidak’s multiple comparison test, ** p = 0.0015; **** p ≤ 0.0001. ( B) Percentage purity data for cardiomyocytes determined by alpha-actinin staining (green), for R-GECO1.0 by RFP staining (red). Mean ±SD, n = 3 technical replicates. One-way ANOVA with Sidak’s multiple comparison test, **** p ≤ 0.0001. ( C) Targeted hiPSC lines show variations in expression of R-GECO1.0 protein upon differentiation. hiPSC lines, identified by OCT4 staining (green, top row) show high R-GECO1.0 expression (red). Upon differentiation to cardiomyocytes, identified by α-actinin staining (green, bottom row), MYH7 WT/MUT 8 and MYH7 MUT/MUT 15 hiPSC-CMs show lower R-GECO1.0 expression (red). Scale bars = 50 µm. ( D) R-GECO1.0 expression in MYH7 MUT/MUT 15 cardiomyocytes is significantly improved with three passages of hiPSC cell culture in 0.3 µg/ml puromycin and differentiation carried out in media supplemented with puromycin. Mean ±SD, n = 3 technical replicates. Unpaired t-test, *** p = 0.0003.

Figure 4.
Figure 4.. Differentiation towards cardiomyocytes or haematopoietic cells results in silencing of AAVS1-targeted transgene after mesoderm induction.

( A) qRT-PCR performed on hiPSCs targeted at the AAVS1 locus with a doxycycline-inducible HOXA9-T2A-mScarlet construct undergoing cardiomyocyte differentiation. Relative expression to untargeted hiPSCs. Reduced expression of the transgene is observed from day two onwards. ( B) Live imaging of AAVS1-targeted hiPSCs undergoing cardiomyocyte differentiation at different timepoints. mScarlet expression peaks on day 0 and reduces throughout the differentiation, despite repeated doxycycline treatment. Scale bars = 50 µm. ( C) Quantification of mScarlet expression using high content image analysis at different timepoints during cardiomyocyte differentiation. ( D) qRT-PCR performed on hiPSCs targeted at the AAVS1 locus with a doxycycline-inducible HOXA9-T2A-mScarlet construct undergoing haematopoietic differentiation. Relative expression to untargeted hiPSCs. Reduced expression of the transgene is observed from day four onwards. ( E) Live imaging of AAVS1-targeted hiPSCs undergoing haematopoietic differentiation at different timepoints. Scale bars = 50 µm. ( F) Quantification of mScarlet expression using high content image analysis shows peak expression on day two and reduced expression thereafter. Mean ±SD, n = 2 differentiations. One-way ANOVA with Dunnett’s multiple comparison test, ** p = 0.0036; **** p ≤ 0.0001. ( G, H) The mesoderm markers MIXL1 and Brachyury ( T) show peak expression at day two of haematopoietic differentiation.

Figure 5.
Figure 5.. Functional application of AAVS1-targeted R-GECO1.0 expressing clones for in vitro disease modelling of HCM and phenotypic rescue with drug treatment.

( A– C) Representative 25-second confocal laser line scan traces and kymographs for isogenic trio of c. MYH7 C9123T R-GECO1.0 expressing hiPSC-CMs at day 30. Abnormal Ca 2+ transient events (red arrows) increase in frequency with mutation load. x-axis scale bar = 20 µm, y-axis scale bar = 5 seconds. ( D) Ca 2+ transient event detection and quantification showing percentage of Ca 2+ transients deemed abnormal. Data presented as mean ±SD, n = 6 scans across three differentiations. Kruskal-Wallis test with Dunn’s multiple comparison test; ** p = 0.0018, **** p < 0.0001. ( E– F) Representative 25-second confocal laser line scan traces and kymographs for isogenic pair of c. ACTC1 G301A R-GECO1.0 expressing hiPSC-CMs at day 30. Abnormal Ca 2+ transient events (red arrows) increase in frequency with mutation load. x-axis scale bar = 20 µm, y-axis scale bar = 5 seconds. ( G) Box-plot showing % of total Ca 2+ transient events detected deemed abnormal by event detection software. Data presented as mean ±SD, n = 5 scans from three differentiations. Unpaired t-test; * p = 0.0118. ( H– L) Representative line scans and event detection quantification showing a reduction in abnormal Ca 2+ transient events upon treatment with 10 µM ranolazine and 10 µM dantrolene compared to 0.1% DMSO vehicle control. Data presented as mean ±SD, n > 4 cells across minimum of two differentiations. Kruskal-Wallis test with Dunn’s multiple comparison test; ** p = 0.0068. x-axis scale bar = 20 µm, y-axis scale bar = 5 seconds.

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