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

Background: 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 human induced pluripotent stem cell (hiPSC) reporters by using a plasmid-based CRISPR/Cas9 D10A nickase strategy. The first intron of PPP1R12C, also known as the AAVS1 locus, was targeted with constructs expressing either a genetically encoded calcium indicator (R-GECO1.0) or a HOXA9-T2A-mScarlet reporter under the control of a pCAG or an 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 98.9% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after 2 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 could be rescued by continuous puromycin drug selection, which allowed the Ca²⁺ responses associated with hypertrophic cardiomyopathy to be investigated 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