Tools
Tools
Bhagwan, Jamie R, Collins, Emma, Mosqueira, Diogo, Bakar, Mine, Johnson, Benjamin B, Thompson, Alexander, Smith, James G W 
ORCID: https://orcid.org/0000-0003-0427-8678 and Denning, Chris
(2020)
Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.
F1000Research, 8.
ISSN 2046-1402
Preview |
PDF (Proof_Version)
Available under License Creative Commons Attribution. Download (6MB) | Preview |
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.
| Item Type: | Article |
|---|---|
| Additional Information: | Copyright: © 2020 Bhagwan JR et al. |
| Uncontrolled Keywords: | sdg 3 - good health and well-being ,/dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being |
| Faculty \ School: | Faculty of Medicine and Health Sciences > Norwich Medical School |
| UEA Research Groups: | Faculty of Medicine and Health Sciences > Research Groups > Cardiovascular and Metabolic Health Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health |
| Depositing User: | LivePure Connector |
| Date Deposited: | 24 Sep 2020 00:04 |
| Last Modified: | 19 Oct 2023 02:45 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/76995 |
| DOI: | 10.12688/f1000research.19894.2 |
Downloads
Downloads per month over past year
Actions (login required)
 |
View Item |