Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.
Version 3 2020-05-21, 14:27Version 3 2020-05-21, 14:27
Version 2 2019-11-08, 15:11Version 2 2019-11-08, 15:11
Version 1 2019-07-17, 10:08Version 1 2019-07-17, 10:08
Posted on 2020-05-21 - 14:27 authored by Jamie Bhagwan
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
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Bhagwan, Jamie; Collins, Emma; Mosqueira, Diogo; Bakar, Mine; Johnson, Benjamin; Thompson, Alexander; et al. (2019). Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.. figshare. Collection. https://doi.org/10.6084/m9.figshare.c.4573316.v3