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

Bhagwan, Jamie R.; Collins, Emma; Mosqueira, Diogo; Bakar, Mine; Johnson, Benjamin B.; Thompson, Alexander; Smith, James G.W.; Denning, Chris

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 Ca2+ 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.

Citation

Bhagwan, J. R., Collins, E., Mosqueira, D., Bakar, M., Johnson, B. B., Thompson, A., Smith, J. G., & Denning, C. (in press). Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour. F1000Research, 8, Article 1911. https://doi.org/10.12688/f1000research.19894.1

• Journal Article Type: Article
• Acceptance Date: Nov 12, 2019
• Online Publication Date: Nov 12, 2019
• Deposit Date: Jun 10, 2020
• Publicly Available Date: Jun 10, 2020
• Journal: F1000Research
• Publisher: F1000Research
• Peer Reviewed: Peer Reviewed
• Volume: 8
• Article Number: 1911
• Series ISSN: 2046-1402
• DOI: https://doi.org/10.12688/f1000research.19894.1
• Keywords: Human induced pluripotent stem cells; CRISPR/Cas9, stem-cell derived cardiomyocytes, stem-cell derived haematopoietic cells, AAVS1 safe harbour, gene targeting, silencing
• Public URL: https://nottingham-repository.worktribe.com/output/3610737
• Publisher URL: https://f1000research.com/articles/8-1911
• Additional Information: Bhagwan JR, Collins E, Mosqueira D et al. Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2019, 8:1911