CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells

Abstract

Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.

Keywords: CRISPR-Cas9; autologous cell replacement; genome editing; hiPSC.

Figure 1. CRISPR-Cas9 Genome Editing of MYOC

A) Gene diagram of MYOC showing specificity of gRNA. B) Plasmid maps are shown for the CRISPR-Cas9 system expressing GFP and a MYOC homology directed repair (pHDR) construct containing a non-integrating mCherry cassette. C) GFP expression at 24 hours is shown after transfecting hiPSCs with 1400, 1600, 1800, and 2000 ng of pX330-U6-Chimeric_BB-CBh-hSpCas9_CMV-GFP (px330-GFP). D) T7 Endonuclease I assays of CRISPR-Cas9 mediated double stranded breaks. Asterisk notes the location of the cleaved PCR products. This assay varies over replicates so caution is needed when comparing the intensity of the cleaved products. The limit of detection is ~5% (Vouillot et al., 2015) E) Using 1800 ng of plasmid the Lipofectamine Stem Transfection Reagent effectively delivers the pX330-U6-Chimeric_BB-CBh-hSpCas9_CMV-GFP to hiPSC. F) Co-delivery of CRISPR plasmid and MYOC HDR plasmid.

Figure 2. CRISPR-Cas9 Mediated Homologous Recombination of MYOC

A) Gene diagram of homology directed repair construct with an integrating drug selection cassette pHDR⁺ was synthesized for MYOC. B) Human induced pluripotent stem cell line generated from a patient with a mutation in MYOC. C) PCR analysis of CRISPR-corrected clones (P1–P8) and an untreated sample (C) using a forward primer targeting SV40pA and a reverse primer unique to the genome. A positive PCR result excludes random genomic incorporation of pHDR⁺. D) T7 Endonuclease I assay screening for clonal purity by indirectly detecting differences in the genome of cells at the guide sequence. Clones P1, P3, P6 and P8 contain at least 95% homogeneity.

Figure 3. CRISPR-Cas9 Mediated Homologous Recombination of RPGR

A) Gene diagram of a homology directed repair construct (pHDR^+/−) for CRISPR correction of RPGR. B–C) gDNA analysis; lane1: Ladder lane 2: CRISPR-mediated HR without positive selection, lane 3: CRISPR-mediated HR with positive selection, Lane 4: CRISPR-mediated HR with dual selection, Lane 5: ddH20 control. B) PCR amplification using a forward primer targeting the SV40 polyadenylation sequence and Reverse Primer 1, which is located only in the genome, shows elimination of the selection cassette via negative selection. C) PCR break point analysis using Forward 1 and Reverse 1 demonstrates Cre-mediated excision of the selection cassette. The amplification in lane 2 is due to the presence of heterogeneous outcomes before selection. (D–E) cDNA analysis; lane1: Ladder, lane2: Untreated, lane3: CRISPR-mediated HR with positive selection, lane4: CRISPR-mediated HR with dual selection. D) rt-PCR analysis of a housekeeping gene, POL2RA, shows robust amplification which excludes the possibility of degraded cDNA. E) rt-PCR analysis of RPGR transcripts during the dual selection process demonstrates RPGR expression after dual selection.