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, 15 (2), 215-226

An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome Editing in Human Pluripotent Stem Cells

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An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome Editing in Human Pluripotent Stem Cells

Federico González et al. Cell Stem Cell.

Abstract

Human pluripotent stem cells (hPSCs) offer a unique platform for elucidating the genes and molecular pathways that underlie complex traits and diseases. To realize this promise, methods for rapid and controllable genetic manipulations are urgently needed. By combining two newly developed gene-editing tools, the TALEN and CRISPR/Cas systems, we have developed a genome-engineering platform in hPSCs, which we named iCRISPR. iCRISPR enabled rapid and highly efficient generation of biallelic knockout hPSCs for loss-of-function studies, as well as homozygous knockin hPSCs with specific nucleotide alterations for precise modeling of disease conditions. We further demonstrate efficient one-step generation of double- and triple-gene knockout hPSC lines, as well as stage-specific inducible gene knockout during hPSC differentiation. Thus the iCRISPR platform is uniquely suited for dissection of complex genetic interactions and pleiotropic gene functions in human disease studies and has the potential to support high-throughput genetic analysis in hPSCs.

Figures

Figure 1
Figure 1. Engineering an iCRISPR platform through generating iCas9 hPSCs
(A) Schematic comparison of the current genome editing approach in hPSCs by electroporation of a ~10 kilobases (kb) CRISPR/Cas9 vector (upper panel) with the iCRISPR platform (lower panel) for genome editing in hPSCs. Cas9 protein (green) binds a chimeric gRNA composed of a constant transactivating structural region (purple) and a variable DNA recognition site (red). The Cas9/gRNA complex binds to DNA and induces a DSB (yellow). TRE, tetracycline response element; CAG, constitutive synthetic promoter; M2rtTA, reverse tetracycline transactivator sequence and protein; doxycycline, red dots. (B) Generation of iCas9 hPSCs through TALEN-mediated gene targeting at the AAVS1 locus. Red lines indicate homology to PPP1R12C intron 1; SA, splice acceptor; 2A, self-cleaving 2A peptide; Puro, Puromycin resistance gene; Neo, Neomycin resistance gene. (C) Southern blot analysis of HUES8 iCas9 lines using 3′ external and 5′ internal probes. Lines carrying desired targeted insertions of the Puro-Cas9 and Neo-M2rtTA donor sequences without random integrations are indicated in red. (D) Quantitative real-time RT-PCR (qRT-PCR) analysis of Cas9 transcript levels with or without doxycycline treatment in HUES8 iCas9 lines. See also Figure S1 and Table S1.
Figure 2
Figure 2. Single and multiplexed gRNA transfection efficiently induces Indels in iCas9 hPSCs
(A) Schematic representation of the experimental procedure. (B) T7EI assay for Cas9-mediated cleavage in HUES8 iCas9 cells using single gRNAs targeting NGN3 (Cr5, 6), GATA6 (Cr1, 8), TET1 (Cr2, 3), TET2 (Cr3, 4), and TET3 (Cr2, 4). All gRNAs were transfected twice, except for GATA6 (Cr1, 8), which were transfected once. In all figures in this study red asterisks indicate the expected T7EI-specific fragments used to quantify Indel frequency (blue). (C) Schematic of Cas9/gRNA-targeting sites (pink arrows in all figures in this study) in TET1 and TET2 loci showing exon structure (blue boxes in all figures in this study), PCR amplicons (light grey boxes in all figures in this study) and StyI or PstI restriction sites used for RFLP analysis. gRNA-targeting sequences, bold; protospacer-adjacent motif (PAM) sequence, orange; Cas9 cleavage site (blue arrow heads); restriction sites, underlined, as in all figures in this study. (D) RFLP analysis upon TET1 (Cr2, 3), TET2 (Cr3, 4) or multiplexed TET1, 2, 3 (Triple) gRNA transfection. In all figures in this study green asterisks indicate uncut PCR fragment used to quantify Indel frequency by RFLP (blue). (E) T7EI assay in HUES8 iCas9 cells transfected with multiplexed GATA4,6 (Double) or TET1, 2, 3 (Triple) gRNAs. See also Figure S2.
Figure 3
Figure 3. Single and multiplexed gene targeting
(A) Strategy of gene targeting. (B and C) Allelic sequence distribution in HUES8 clonal lines generated with single gRNAs targeting NGN3 (Cr5, 6), GATA4 (Cr2), GATA6 (Cr1, 8), TET1 (Cr2), TET2 (Cr4), TET3 (Cr4) (B) or multiplexed gRNAs targeting GATA4 and 6 (Cr2, Cr1 respectively) or TET1, 2 and 3 (Cr2, Cr4, Cr4, respectively) (C). (D) Representative sequences of various knockout (KO) mutant clones with PAM sequences labeled in red. pm: point mutation. (E) Analysis of 5hmC levels in DNA isolated from TET1, 2, 3 triple-targeted hESC clones by dot blot assay using an anti-5hmC antibody. Wild-type HUES8 iCas9 cells and human foreskin fibroblasts (HFF) are used as controls. See also Figure S3, Table S2, S3.
Figure 4
Figure 4. HDR-mediated genome editing
(A) Schematic of Cas9/gRNA and ssDNA oligo targeting sites at the GATA6 locus. A C>T substitution (green) was introduced in the ssDNA HDR template, generating a new BsgI restriction site (underlined), resulting in an R456C amino acid substitution in GATA6. (B) Strategy of HDR-mediated genome editing. (C and D) T7EI and RFLP assay in HUES8 (C) and BJ iPSCs (D) cotransfected with GATA6 gRNA (Cr8) and ssDNA HDR repair template. (E and F) RFLP analysis (E) and allelic sequence distribution (F) in clones generated with GATA6 gRNA/ssDNA. wt, wild-type; mut, mutation. “R456C + random mut” includes clones with undesired mutations in addition to the R456C modification in one or both alleles. (G) Representative sequences of one homozygous (R456C/ R456C) and one compound heterozygous (R456C/R456C + additional mutations) GATA6 mutant clone.
Figure 5
Figure 5. Generation of an allelic series at the APOE locus
(A) Sequence analysis of SNP rs429358 in parental HUES8 hESCs and BJ hiPSCs lines (ε3/ε3) and derived HDR-mediated edited clones (ε3/ε4 or ε4/ε4). Ratios indicate the number of colonies with the specified genotype out of the total number of colonies analyzed. (B) Schematic of Cas9/gRNA and ssDNA oligo targeting sites at the APOE locus. A T(red)>C(blue) substitution was introduced in the ε4 ssDNA to convert the ε3 allele into ε4, in addition a C>G substitution (green) was introduced generating a novel BanI, and disrupting the endogenous NotI restriction site (underlined). SNP rs429358 and BanI sites are indicated in APOE exon 4. (C and D) T7EI and RFLP assay in HUES8 hESCs (C) and BJ iPSCs (D) cotransfected with APOE gRNA (Cr3) and ε4 ssDNA HDR repair template. Ratios indicate the number colonies with the specified genotype out of the total number of colonies analyzed. See also Figure S4.
Figure 6
Figure 6. Inducible gene knockout
(A) Strategy of inducible gene knockout. DE, definitive endoderm stage; PP pancreatic progenitor stage. (B and C) T7EI assay in HUES8 iCas9 cells transfected with gRNAs targeting NGN3 (Cr6) at the PP stage (B), or sorted CXCR4+ DE cells differentiated to the PP stage, and transfected with gRNAs targeting NGN3 (Cr6), GATA6 (Cr8), TET1 (Cr2), TET2 (Cr4) and TET3 (Cr4) (C). (D) RFLP analysis of TET1 Cr2 and TET2 Cr4 transfected samples from (C). (E) Allelic sequence distribution in differentiated HUES8 iCas9 cells transfected with different gRNAs at PP stage. wt, wild-type; mut, mutation; FS, frameshift. (F) Schematic illustrating the TALEN-mediated establishment of iCr hESCs for inducible gene knockout. A U6 Pol III driving constitutive expression of a specific gRNA is included 3′ of the inducible Cas9 expression cassette at the AAVS1 locus, allowing gene knockout in all doxycycline treated iCr hESCs. (G) T7EI assay in differentiated iCr hESC lines expressing NGN3-Cr5 (#7), Cr6 (#12) or TET2-Cr4 (#2) gRNAs, treated with doxycycline at the PP stage. (H) RFLP analysis of iCrTET2 (Cr4, #2) hESCs from (G). (I) Allelic sequence distribution in differentiated iCr hESCs treated with doxycycline at PP stage. The number of independent clones analyzed is indicated above each column in panel E and I. See also Figure S5.

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