Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives

Abstract

The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

“…This allowed us to confirm GFP-tag incorporation without large insertions or deletions (Figure 2A, middle panel, step 2) (Jasin and Rothstein, 2013; Oceguera-Yanez et al. , 2016).…”

“…Donor plasmid integration was the most common category of imprecise editing, affecting 45% of all clones (Figure 2F). Our data suggest that this frequently occurs at the edited locus as a faulty by-product of the editing process and that screening by junctional PCR alone, without a method to directly detect the plasmid backbone, leads to misidentification of clones with imprecise editing, despite appropriate localization of the tagged protein resulting from the edit (Figure 2F and Supplemental Figure S4; Jasin and Rothstein, 2013; Oceguera-Yanez et al. , 2016).…”

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…11 Genes introduced into the AAVS1 site in human PSCs showed stable expression after in vitro and in vivo differentiation. 5,[12][13][14][15] The clustered regularly interspaced short palindromic www.moleculartherapy.org repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is a highly efficient RNA-guided DNA nuclease system modified from an adaptive immune system of bacteria and archaea to allow sequence-specific genome editing in human cells. [16][17][18] Recent reports have indicated that the CRISPR/Cas9 system can be used for genetic modification in NHPs; however, this approach has not yet been applied to insert genes of interest into the AAVS1 safe harbor in NHP PSCs.…”

Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

“…To further test our miR-Cas9 switch in different cell types, we next chose a human iPS cell line (201B7) that contains a single copy of the EGFP gene at the AAVS1 locus, which is often considered a safe-harbor for transgene targeting (hiPSC-EGFP) (19). We used the miR-302-Cas9 switch to selectively repress Cas9 activity in hiPSCs.…”

“…The heterozygously targeted AAVS1-CAG::GFP human iPSCs 317-12 line (hiPSC-EGFP) (19) was maintained in StemFit (Ajinomoto) on laminin-511 E8 (iMatrix-511, nippi). HeLa-EGFP cells were cultured in DMEM High Glucose (nacalai tesque) supplemented with 10% FBS (JBS) and Hygromycin B (50 μg/ml).…”